Photoreactive regulator of protein function and methods of use thereof

ABSTRACT

The present invention provides a synthetic regulator of protein function, which regulator is a light-sensitive regulator. The present invention further provides a light-regulated polypeptide that includes a subject synthetic regulator. Also provided are cells and membranes comprising a subject light-regulated polypeptide. The present invention further provides methods of modulating protein function, involving use of light. The present invention further provides methods of identifying agents that modulate protein function.

CROSS-REFERENCE

This application claims the benefit of U.S. Provisional PatentApplication No. 60/737,935, filed Nov. 18, 2005, which application isincorporated herein by reference in its entirety.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH

This invention was made with Government support under contract MH60711awarded by the National Institutes of Health. The Government has certainrights in this invention.

BACKGROUND OF THE INVENTION

The precise regulation of protein activity is fundamental to life. Amechanism of regulation, found across protein classes, from enzymes, tomotors, to signaling proteins, is allosteric control of an active siteby a remote regulatory binding site.

Many proteins function like molecular machines that undergo mechanicalmovements in response to input signals. These signals can consist ofchanges in voltage, membrane tension, temperature or, most commonly,ligand concentration. Ligands provide information about events in theexternal world, or about the energetic or biosynthetic state of thecell, and can be as small as a proton or as large as a whole protein. Inallostery, ligand binding induces a structural change of a sensordomain, which propagates to a functional domain of the protein andalters its behavior. Such conformational control can operate over longdistances, crossing a membrane or passing from one protein to another ina complex.

There is a need in the art for methods of regulating protein function.The present invention addresses this need.

LITERATURE

-   Lester et al. J. Gen. Physiol. 75, 207-232 (1980); Banghart et al.    Nature Neurosci. 7, 1381-1386 (2004).

SUMMARY OF THE INVENTION

The present invention provides a synthetic regulator of proteinfunction, which regulator is a light-sensitive (photoreactive)regulator. The present invention further provides a light-regulatedpolypeptide that includes a subject synthetic regulator. Also providedare cells and membranes comprising a subject light-regulatedpolypeptide. The present invention further provides methods ofmodulating protein function, involving use of light. The presentinvention further provides methods of identifying agents that modulateprotein function.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A and 1B depict photoisomerization of MAL-AZO-QA gates ioniccurrents through modified Shaker channels.

FIGS. 2A-D depict photocontrol of MAL-AZO-QA-modified Shaker channels inXenopus oocytes.

FIGS. 3A-D depict absorbance and action spectra of MAL-AZO-QA.

FIGS. 4A-C depict expression of light-activated channels conferslight-sensitivity on hippocampal pyramidal neurons.

FIGS. 5A-C schematically depict a design of an allosteric photoswitch.

FIGS. 6A-D depict structures and fit of photoswitched agonist and iGluR6LBD. (a) Chemical structure of the iGluR6 agonists (2S,4R)-4-methylglutamate 1, (2S,4R)-4-allyl glutamate 2 and tether model 3.

FIGS. 7A-E depict calcium imaging of iGluR6 activity.

FIG. 8 depicts a scheme for total synthesis of MAG 4.

FIGS. 9A and 9B depict whole-cell patch-clamp current recordings fromHEK293 cells expressing iGluR6-L439C after conjugation of MAG 4.

FIGS. 10A and 10 B depict modular photoswitchable tethered ligands.

FIGS. 11A-E depict photostationary state determination by NMR andspectral sensitivity of photoresponses.

FIGS. 12A and 12B depict thermal relaxation of MAG.

FIGS. 13A-D depict MAG-1 conjugation to iGluR6-L439C by affinitylabeling.

FIG. 14 depicts a patch clamp trace showing that MAG functions as a fullagonist.

FIGS. 15A-D depict results showing that the effective localconcentration of MAG-1 is ˜12 mM.

FIGS. 16A-D depict tether length dependence on channel activation.

FIGS. 17A-D depict the photoswitchable affinity label (PAL) approach togenerating light-regulated ion channels.

FIGS. 18A-D depict photocontrol of wild-type K⁺ channels.

FIGS. 19A-C depict photocontrol of K⁺ current in cultured hippocampalneurons.

FIGS. 20A and 20B depict regulation of action potential firing withlight. The “□” symbols in FIG. 20B are short bars, and represent pulsesof light.

FIG. 21 depicts neuronal survival after PAL treatment.

FIGS. 22A-C depict photocontrol of action potential firing in cerebellarslices.

FIGS. 23A-D depict trains of action potential firing followingphotostimulation.

FIGS. 24A and 24B depict dependence of speed of gating on illuminationintensity.

FIGS. 25A-C depict designed temporal firing patterns.

FIG. 26 depicts photo-stimulation of LiGluR up to at variousfrequencies.

FIGS. 27A-C depict wavelength-dependent depolarization.

FIG. 28 depicts sustained firing at intrinsic frequency of the cellusing brief pulses of illumination followed by dark.

FIGS. 29A and 29B depict retcam images of rat eyes injected with arecombinant adeno-associated virus (rAAV) vector comprising a nucleotidesequence encoding a potassium channel-green fluorescent protein(SPARK-GFP) fusion protein under the control of a synapsin promoter(AAV-SYN-SPARK-GFP).

FIG. 30 depicts a flatmount of a rat eye injected withAAV-SYN-SPARK-GFP.

FIG. 31 depicts in vivo expression of SPARK.

FIGS. 32A and 32B depict inhibition by light of spontaneous firing ofSPARK-expressing retinal ganglion cells in intact retina.

FIGS. 33A and 33B depict light sensitivity imparted on rat retina byPAL.

DEFINITIONS

The terms “peptide,” “polypeptide,” and “protein” are usedinterchangeably herein, and refer to a polymeric form of amino acids ofany length, which can include coded and non-coded amino acids,chemically or biochemically modified or derivatized amino acids, andpolypeptides having modified peptide backbones. The term includes fusionproteins, including, but not limited to, fusion proteins with aheterologous amino acid sequence, fusions with heterologous andhomologous leader sequences, with or without N-terminal methionineresidues; immunologically tagged proteins; and the like. The term“polypeptide” includes polypeptides comprising one or more of a fattyacid moiety, a lipid moiety, a sugar moiety, and a carbohydrate moiety.The term “polypeptides” includes post-translationally modifiedpolypeptides.

The term “naturally-occurring” as used herein as applied to apolypeptide, a cell, or an organism, refers to a polypeptide, cell, ororganism that is found in nature. For example, a polypeptide having anamino acid sequence that is present in an organism (including viruses)that can be isolated from a source in nature and which has not beenintentionally modified by a human in the laboratory is naturallyoccurring.

The term “determining” includes any form of measurement, and includesdetermining if an effect is present or not. The terms “determining”,“measuring”, “evaluating”, “assessing” and “assaying” are usedinterchangeably and include quantitative and qualitative determinations.Assessing may be relative or absolute. “Determining the effect of”includes determining the degree of an effect, and/or determining whetherany effect has occurred. As used herein, the terms “determining,”“measuring,” and “assessing,” and “assaying” are used interchangeablyand include both quantitative and qualitative determinations.

A “host cell,” or “a cell,” as used herein, denotes an in vivo or invitro prokaryotic cell, an in vivo or in vitro eukaryotic cell, or acell from a multicellular organism (e.g., a cell line) cultured in vitroas a unicellular entity. A cell includes a cell that comprises a subjectlight-regulated polypeptide. A “host cell” includes cells that can be,or have been, used as recipients for a subject synthetic regulator. A“host cell” includes cells that can be, or have been, used as recipientsfor an exogenous nucleic acid. It is understood that the progeny of asingle cell may not necessarily be completely identical in morphology orin genomic or total DNA complement as the original parent, due tonatural, accidental, or deliberate mutation. A “recombinant host cell”(also referred to as a “genetically modified host cell”) is a host cellinto which has been introduced a heterologous nucleic acid, e.g., anexpression vector. For example, in some embodiments a subject host cellis a genetically modified eukaryotic host cell, by virtue ofintroduction into a suitable eukaryotic host cell a heterologous nucleicacid, e.g., an exogenous nucleic acid that is foreign to the eukaryotichost cell, or a recombinant nucleic acid that is not normally found inthe eukaryotic host cell.

As used herein the term “isolated” is meant to describe apolynucleotide, a polypeptide, or a cell that is in an environmentdifferent from that in which the polynucleotide, the polypeptide, or thecell naturally occurs. An isolated genetically modified host cell may bepresent in a mixed population of genetically modified host cells.

The term “conservative amino acid substitution” refers to theinterchangeability in proteins of amino acid residues having similarside chains. For example, a group of amino acids having aliphatic sidechains consists of glycine, alanine, valine, leucine, and isoleucine; agroup of amino acids having aliphatic-hydroxyl side chains consists ofserine and threonine; a group of amino acids having amide-containingside chains consists of asparagine and glutamine; a group of amino acidshaving aromatic side chains consists of phenylalanine, tyrosine, andtryptophan; a group of amino acids having basic side chains consists oflysine, arginine, and histidine; and a group of amino acids havingsulfur-containing side chains consists of cysteine and methionine.Exemplary conservative amino acids substitution groups are:valine-leucine-isoleucine, phenylalanine-tyrosine, lysine-arginine,alanine-valine, and asparagine-glutamine.

As used herein, the terms “treatment,” “treating,” and the like, referto obtaining a desired pharmacologic and/or physiologic effect. Theeffect may be prophylactic in terms of completely or partiallypreventing a disease or symptom thereof and/or may be therapeutic interms of a partial or complete cure for a disease and/or adverse affectattributable to the disease. “Treatment,” as used herein, covers anytreatment of a disease in a mammal, particularly in a human, andincludes: (a) preventing the disease or a symptom of a disease fromoccurring in a subject which may be predisposed to the disease but hasnot yet been diagnosed as having it (e.g., including diseases that maybe associated with or caused by a primary disease; (b) inhibiting thedisease, i.e., arresting its development; and (c) relieving the disease,i.e., causing regression of the disease.

The terms “individual,” “host,” “subject,” and “patient” are usedinterchangeably herein, and refer to a mammal, including, but notlimited to, primates, including simians and humans; rodents, includingrats and mice; bovines; equines; ovines; felines; canines; and the like.“Mammal” means a member or members of any mammalian species, andincludes, by way of example, canines; felines; equines; bovines; ovines;rodentia, etc. and primates, particularly humans. Non-human animalmodels, particularly mammals, e.g. non-human primates, murines,lagomorpha, etc. may be used for experimental investigations.

A “therapeutically effective amount” or “efficacious amount” means theamount of a compound that, when administered to a mammal or othersubject for treating a disease, condition, or disorder, is sufficient toeffect such treatment for the disease, condition, or disorder. The“therapeutically effective amount” will vary depending on the compound,the disease and its severity and the age, weight, etc., of the subjectto be treated.

The term “unit dosage form,” as used herein, refers to physicallydiscrete units suitable as unitary dosages for human and animalsubjects, each unit containing a predetermined quantity of a compound(e.g., an aminopyrimidine compound, as described herein) calculated inan amount sufficient to produce the desired effect in association with apharmaceutically acceptable diluent, carrier or vehicle. Thespecifications for unit dosage forms depend on the particular compoundemployed and the effect to be achieved, and the pharmacodynamicsassociated with each compound in the host.

A “pharmaceutically acceptable excipient,” “pharmaceutically acceptablediluent,” “pharmaceutically acceptable carrier,” and “pharmaceuticallyacceptable adjuvant” means an excipient, diluent, carrier, and adjuvantthat are useful in preparing a pharmaceutical composition that aregenerally safe, non-toxic and neither biologically nor otherwiseundesirable, and include an excipient, diluent, carrier, and adjuvantthat are acceptable for veterinary use as well as human pharmaceuticaluse. “A pharmaceutically acceptable excipient, diluent, carrier andadjuvant” as used in the specification and claims includes both one andmore than one such excipient, diluent, carrier, and adjuvant.

As used herein, a “pharmaceutical composition” is meant to encompass acomposition suitable for administration to a subject, such as a mammal,especially a human. In general a “pharmaceutical composition” issterile, and preferably free of contaminants that are capable ofeliciting an undesirable response within the subject (e.g., thecompound(s) in the pharmaceutical composition is pharmaceutical grade).Pharmaceutical compositions can be designed for administration tosubjects or patients in need thereof via a number of different routes ofadministration including oral, buccal, rectal, parenteral,intraperitoneal, intradermal, intracheal, intramuscular, subcutaneous,and the like.

Before the present invention is further described, it is to beunderstood that this invention is not limited to particular embodimentsdescribed, as such may, of course, vary. It is also to be understoodthat the terminology used herein is for the purpose of describingparticular embodiments only, and is not intended to be limiting, sincethe scope of the present invention will be limited only by the appendedclaims.

Where a range of values is provided, it is understood that eachintervening value, to the tenth of the unit of the lower limit unlessthe context clearly dictates otherwise, between the upper and lowerlimit of that range and any other stated or intervening value in thatstated range, is encompassed within the invention. The upper and lowerlimits of these smaller ranges may independently be included in thesmaller ranges, and are also encompassed within the invention, subjectto any specifically excluded limit in the stated range. Where the statedrange includes one or both of the limits, ranges excluding either orboth of those included limits are also included in the invention.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Although any methods andmaterials similar or equivalent to those described herein can also beused in the practice or testing of the present invention, the preferredmethods and materials are now described. All publications mentionedherein are incorporated herein by reference to disclose and describe themethods and/or materials in connection with which the publications arecited.

It must be noted that as used herein and in the appended claims, thesingular forms “a,” “and,” and “the” include plural referents unless thecontext clearly dictates otherwise. Thus, for example, reference to “asynthetic regulator” includes a plurality of such regulators andreference to “the light-regulated polypeptide” includes reference to oneor more light-regulated polypeptides and equivalents thereof known tothose skilled in the art, and so forth. It is further noted that theclaims may be drafted to exclude any optional element. As such, thisstatement is intended to serve as antecedent basis for use of suchexclusive terminology as “solely,” “only” and the like in connectionwith the recitation of claim elements, or use of a “negative”limitation.

The publications discussed herein are provided solely for theirdisclosure prior to the filing date of the present application. Nothingherein is to be construed as an admission that the present invention isnot entitled to antedate such publication by virtue of prior invention.Further, the dates of publication provided may be different from theactual publication dates which may need to be independently confirmed.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides a synthetic regulator of proteinfunction, which regulator is a light-sensitive regulator. The presentinvention further provides a light-regulated polypeptide that includes asubject synthetic regulator. Also provided are cells and membranescomprising a subject light-regulated polypeptide. The present inventionfurther provides methods of modulating protein function, involving useof light. The present invention further provides methods of identifyingagents that modulate protein function.

Synthetic Regulator of Protein Function

The present invention provides a synthetic regulator of proteinfunction, light-regulated polypeptides comprising the regulator, anddevices comprising the polypeptides. A subject synthetic proteinregulator comprises: a) a linker domain, comprising a moiety thatprovides for covalent linkage to an amino acid side chain; b) aphotoisomerizable group; and c) a ligand that binds to a ligand bindingsite (e.g., an active site, an allosteric site, a pore of an ionchannel, etc.) of a protein. A subject synthetic protein regulator (alsoreferred to as a “synthetic regulator,” or “a photoswitch”) is suitablefor attachment to a variety of polypeptides, includingnaturally-occurring (native, or endogenous) polypeptides, recombinantpolypeptides, synthetic polypeptides, etc.

A subject synthetic regulator can be provided in any number ofconfigurations, including linear and branched. In some embodiments, asubject synthetic regulator has the structure: (A)_(n)-(B)_(m)-(C)_(p),where A is a linker domain, B is a photoisomerizable group, and C is aligand, and where each of n, m, and p is independently 1 to 10, e.g.,where each of n, m, and p is independently one, two, three, four, five,six, seven, eight, nine, or ten. In some embodiments, each of n, m, andp is 1, e.g., a subject synthetic regulator has the structure A-B-C. Inother embodiments, a subject synthetic regulator comprises two or moredifferent linker domains. In other embodiments, a subject syntheticregulator comprises two or more (e.g., 2 to 10, e.g., two, three, four,five, six, seven, eight, nine, or ten) photoisomerizable groups. In someembodiments, where the synthetic regulator comprises two or morephotoisomerizable groups, the two or more photoisomerizable groups arearranged in tandem, either directly or separated by a spacer. In someembodiments, a subject synthetic regulator comprises a single linkerdomain, and two or more photoisomerizable groups, arranged in tandem,either directly or separated by a spacer. In other embodiments, asubject synthetic regulator comprises two or more different ligands.

In other embodiments, a subject synthetic regulator has the structure:C-X₁(A)-B-X₂(A)-C, where A is a linker domain, B is a photoisomerizablegroup, and C is a ligand, where X₁, when present, is a spacer, where X₂,when present, is a spacer, and where X(A) indicates that A branches offof X. Suitable spacers include peptide spacers (e.g., spacers of fromabout 1 to about 20 amino acids in length; non-peptide spacers, e.g.,non-peptide polymers of various numbers of monomeric units, e.g., fromone to about 20 units. In these embodiments, B can be present inmultiple copies, either directly or in tandem.

Photoisomerizable Group

The photoisomerizable group is one that changes from a first isomericform to a second isomeric form upon exposure to light of differentwavelengths, or upon a change in exposure from dark to light, or fromlight to dark. For example, in some embodiments, the photoisomerizablegroup is in a first isomeric form when exposed to light of a firstwavelength, and is in a second isomeric form when exposed to light of asecond wavelength. Suitable photoisomerizable groups includestereoisomers and constitutional isomers.

The first wavelength and the second wavelength can differ from oneanother by from about 1 nm to about 2000 nm or more, e.g., from about 1nm to about 10 nm, from about 10 nm to about 20 nm, from about 20 nm toabout 50 nm, from about 50 nm to about 75 nm, from about 75 nm to about100 nm, from about 100 nm to about 125 nm, from about 125 nm to about150 nm, or from about 150 nm to about 200 nm, from about 200 nm to about500 nm, from about 500 nm to about 800 nm, from about 800 nm to about1000 nm, from about 1000 nm to about 1500 nm, from about 1500 nm toabout 2000 nm, or more than 2000 nm.

In other embodiments, the photoisomerizable group is in a first isomericform when exposed to light of a wavelength λ₁, and is in a secondisomeric form in the absence of light (e.g., in the absence of light,the photoisomerizable group undergoes spontaneous relaxation into thesecond isomeric form). In these embodiments, the first isomeric form isinduced by exposure to light of wavelength λ₁, and the second isomericform is induced by not exposing the photoisomerizable group to light,e.g., keeping the photoisomerizable group in darkness. In otherembodiments, the photoisomerizable group is in a first isomeric form inthe absence of light, e.g., when the photoisomerizable group is in thedark; and the photoisomerizable group is in a second isomeric form whenexposed to light of a wavelength λ₁. In other embodiments, thephotoisomerizable group is in a first isomeric form when exposed tolight of a first wavelength λ₁, and the photoisomerizable group is in asecond isomeric form when exposed to light of second wavelength λ₂.

For example, in some embodiments, the photoisomerizable group is in atrans configuration in the absence of light, or when exposed to light ofa first wavelength; and the photoisomerizable group is in a cisconfiguration when exposed to light, or when exposed to light of asecond wavelength that is different from the first wavelength. Asanother example, in some embodiments, the photoisomerizable group is ina cis configuration in the absence of light, or when exposed to light ofa first wavelength; and the photoisomerizable group is in a transconfiguration when exposed to light, or when exposed to light of asecond wavelength that is different from the first wavelength.

The wavelength of light that effects a change from a first isomeric formto a second isomeric form ranges from 10⁻⁸ m to about 1 m, e.g., fromabout 10⁻⁸ m to about 10⁻⁷ m, from about 10⁻⁷ m to about 10⁻⁶ m, fromabout 10⁻⁶ m to about 10⁻⁴ m, from about 10⁻⁴ m to about 10⁻² m, or fromabout 10⁻² m to about 1 m. “Light,” as used herein, refers toelectromagnetic radiation, including, but not limited to, ultravioletlight, visible light, infrared, and microwave.

The wavelength of light that effects a change from a first isomeric formto a second isomeric form ranges in some embodiments from about 200 nmto about 800 nm, e.g., from about 200 nm to about 250 nm, from about 250nm to about 300 nm, from about 300 nm to about 350 nm, from about 350 nmto about 400 nm, from about 400 nm to about 450 nm, from about 450 nm toabout 500 nm, from about 500 nm to about 550 nm, from about 550 nm toabout 600 nm, from about 600 nm to about 650 nm, from about 650 nm toabout 700 nm, from about 700 nm to about 750 nm, or from about 750 nm toabout 800 nm, or greater than 800 nm.

In other embodiments, the wavelength of light that effects a change froma first isomeric form to a second isomeric form ranges from about 800 nmto about 2500 nm, e.g., from about 800 nm to about 900 nm, from about900 nm to about 1000 nm, from about 1000 nm to about 1200 nm, from about1200 nm to about 1400 nm, from about 1400 nm to about 1600 nm, fromabout 1600 nm to about 1800 nm, from about 1800 nm to about 2000 nm,from about 2000 nm to about 2250 nm, or from about 2250 nm to about 2500nm. In other embodiments, the wavelength of light that effects a changefrom a first isomeric form to a second isomeric form ranges from about 2nm to about 200 nm, e.g., from about 2 nm to about 5 nm, from about 5 nmto about 10 nm, from about 10 nm to about 25 nm, from about 25 nm toabout 50 nm, from about 50 nm to about 75 nm, from about 100 nm, fromabout 100 nm to about 150 nm, or from about 150 nm to about 200 nm.

The difference between the first wavelength and the second wavelengthcan range from about 1 nm to about 2000 nm or more, as described above.Of course, where the synthetic light regulator is switched from darknessto light, the difference in wavelength is from essentially zero to asecond wavelength.

The intensity of the light can vary from about 1 W/m² to about 50 W/m²,e.g., from about 1 W/m² to about 5 W/m², from about 5 W/m² to about 10W/m², from about 10 W/m², from about 10 W/m² to about 15 W/m², fromabout 15 W/m² to about 20 W/m², from about 20 W/m² to about 30 W/m²,from about 30 W/m² to about 40 W/m², or from about 40 W/m² to about 50W/m². The intensity of the light can vary from about 1 μW/cm² to about100 μW/cm², e.g., from about 1 μW/cm² to about 5 μW/cm², from about 5μW/cm² to about 10 μW/cm², from about 10 μW/cm² to about 20 μW/cm², fromabout 20 μW/cm² to about 25 μW/cm², from about 25 μW/cm² to about 50μW/cm², from about 50 μW/cm² to about 75 μW/cm², or from about 75 μW/cm²to about 100 μW/cm². In some embodiments, the intensity of light variesfrom about 1 μW/mm² to about 1 W/mm², e.g., from about 1 μW/mm² to about50 μW/mm², from about 50 μW/mm² to about 100 μW/mm², from about 100μW/mm² to about 500 μW/mm², from about 500 μW/mm² to about 1 mW/mm²,from about 1 mW/mm² to about 250 mW/mm², from about 250 mW/mm² to about500 mW/mm², or from about 500 mW/mm² to about 1 W/mm².

In some embodiments, the change from a first isomeric form to a secondisomeric form of the photoisomerizable group is effected using sound,instead of electromagnetic (EM) radiation (light). For example, in someembodiments, the change from a first isomeric form to a second isomericform of the photoisomerizable group is effected using ultrasound.

Photoisomerizable groups are known in the art, and any knownphotoisomerizable group can be included in a subject synthetic regulatorof protein function. Suitable photoisomerizable groups include, but arenot limited to, azobenzene and derivatives thereof; spiropyran andderivatives thereof; triphenyl methane and derivatives thereof;4,5-epoxy-2-cyclopentene and derivatives thereof; fulgide andderivatives thereof; thioindigo and derivatives thereof; diaryletheneand derivatives thereof; diallylethene and derivatives thereof;overcrowded alkenes and derivatives thereof; and anthracene andderivatives thereof.

Suitable spiropyran derivatives include, but are not limited to,1,3,3-trimethylindolinobenzopyrylospiran;1,3,3-trimethylindolino-6′-nitrobenzopyrylospiran;1,3,3-trimethylindolino-6′-bromobenzopyrylospiran;1-n-decyl-3,3-dimethylindolino-6′-nitrobenzopyrylospiran;1-n-octadecy-1-3,3-dimethylindolino-6′-nitrobenzopyrylospiran;3′,3′-dimethyl-6-nitro-1′-[2-(phenylcarbamoyl)ethyl]spiro;[2H-1-benzopyran-2,2′-indoline];1,3,3-trimethylindolino-8′-methoxybenzopyrylospiran; and1,3,3-trimethylindolino-β-naphthopyrylospiran. Also suitable for use isa merocyanine form corresponding to spiropyran or a spiropyranderivative.

Suitable triphenylmethane derivatives include, but are not limited to,malachite green derivatives. specifically, there can be mentioned, forexample, bis[dimethylamino)phenyl]phenylmethanol,bis[4-(diethylamino)phenyl]phenylmethanol,bis[4-(dibuthylamino)phenyl]phenylmethanol andbis[4-(diethylamino)phenyl]phenylmethane.

Suitable 4,5-epoxy-2-cyclopentene derivatives include, for example,2,3-diphenyl-1-indenone oxide and 2′,3′-dimethyl-2,3-diphenyl-1-indenoneoxide.

Suitable azobenzene compounds include, e.g., compounds having azobenzeneresidues crosslinked to a side chain, e.g., compounds in which4-carboxyazobenzene is ester bonded to the hydroxyl group of polyvinylalcohol or 4-carboxyazobenzene is amide bonded to the amino group ofpolyallylamine. Also suitable are azobenzene compounds having azobenzeneresidues in the main chain, for example, those formed by ester bondingbis(4-hydroxyphenyl)dimethylmethane (also referred to as bisphenol A)and 4,4′-dicarboxyazobenzene or by ester bonding ethylene glycol and4,4′-dicarboxyazobenzene.

Suitable fulgide derivatives include, but are not limited to,isopropylidene fulgide and adamantylidene fulgide.

Suitable diallylethene derivatives include, for example,1,2-dicyano-1,2-bis(2,3,5-trimethyl-4-thienyl)ethane;2,3-bis(2,3,5-trimethyl-4-thiethyl) maleic anhydride;1,2-dicyano-1,2-bis(2,3,5-trimethyl-4-selenyl)ethane;2,3-bis(2,3,5-trimethyl-4-selenyl) maleic anhydride; and1,2-dicyano-1,2-bis(2-methyl-3-N-methylindole)ethane.

Suitable diarylethene derivatives include but are not limited to,substituted perfluorocylopentene-bis-3-thienyls andbis-3-thienylmaleimides.

Suitable overcrowded alkenes include, but are not limited to,cis-2-nitro-7-(dimethylamino)-9-(2′,3′-dihydro-1′H-naphtho[2,1-b]thiopyran-1′-ylidene)-9H-thioxantheneandtrans-dimethyl-[1-(2-nitro-thioxanthen-9-ylidene)-2,3-dihydro-1H-benzo[f]thiochromen-8-yl]amine.Overcrowded alkenes are described in the literature. See, e.g., terWielet al. (2005) Org. Biomol. Chem. 3:28-30; and Geertsema et al. (1999)Agnew CHem. Int. Ed. Engl. 38:2738.

Other suitable photoisomerizable moieties include, e.g., reactive groupscommonly used in affinity labeling, including diazoketones, aryl azides,diazerenes, and benzophenones.

Linker Domain

The linker domain is any of a variety of linkers that provide for stableassociation of the synthetic regulator with a polypeptide. The linkerdomain includes a binding moiety that provides for stable associationwith a polypeptide. In some embodiments, the linker domain provides forstable association with an amino acid side chain in a polypeptide. Inother embodiments, the linker domain provides for stable association ofthe synthetic regulator with a sugar residue in the polypeptide. Inother embodiments, the linker domain provides for stable association ofthe synthetic regulator with a moiety other than a sugar residue or anamino acid side chain. In some embodiments, the linked domain comprisesa reactive electrophile that provides for stable association with anamino acid in the ligand-binding polypeptide. In some embodiments, thelinked domain comprises a reactive electrophile that provides for stableassociation with an amino acid at or near a ligand-binding site in aligand-binding protein.

Stable association of the synthetic regulator with a polypeptideincludes covalent linkage; as well as non-covalent associations such asionic interactions, and the like. In general, where the stableassociation is a non-covalent association, the stable association is ahigh-affinity association, e.g., the stable association between thesynthetic regulator and the polypeptide has an affinity of from about10⁻⁹ M to about 5×10⁻⁹ M, from about 5×10⁻⁹ M to about 10⁻¹⁰ M, fromabout 10⁻¹⁰ M to about 5×10⁻¹⁰ M, from about 5×10⁻¹⁰ M to about 10⁻¹¹ M,from about 5×10⁻¹¹ M to about 10⁻¹² M, or greater. In some embodiments,e.g., where a subject synthetic regulator comprises two or more linkerdomains, each of the linker domains may provide for attachment to apolypeptide with an affinity of less than about 10⁻⁹ M, but together thetwo or more linker domains provide for a binding affinity that is 10⁻⁹ Mor greater.

Covalent Linkage to an Amino Acid Residue

In some embodiments, the linker domain provides for stable association,e.g., a covalent linkage, with an amino acid side chain in apolypeptide. Linkage of the synthetic regulator to a polypeptide can bevia a tyrosine residue, a tryptophan residue, a serine residue, athreonine residue, cysteine residue, a histidine residue, an arginineresidue, a lysine residue, an aspartic acid residue, a glutamic acidresidue, or any natural or unnatural amino acid in the polypeptide thatis accessible for reacting with the linker domain of the syntheticregulator. Suitable binding moieties include, but are not limited to, amaleimide, an acrylic ester, an acrylic amide (an acrylamide), anα-haloacetamide, an epoxide, an O-succinimidyl ester, a disulfide, and amethanethiosulfonate compound. In some embodiments, the binding moietyis other than a bromomethyl moiety, e.g, in some embodiments, abromomethyl moiety is specifically excluded. For examples of covalentlinkage to an amino acid residue see e.g. Hermanson (1996) BioconjugateTechniques, Academic Press.

Where the amino acid to which the synthetic regulator is to be linked isa cysteine residue, the linker domain will comprise a moiety such as,e.g, a vinylsulfone group, maleimide; a substituted maleimide, such asmaleic anhydride; orthopyridyl-disulfide; a methanethiosulfonate; adisulfide; and the like. Where the amino acid to which the syntheticregulator is to be linked is a lysine residue, the linker domain will insome embodiments comprise a moiety such as, e.g, carbodiimide EDC(1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride). Where theamino acid to which the synthetic regulator is to be linked is anarginine residue, the linker domain will comprise, e.g, 2,3-butanedione,phenylglyoxal, or glyoxal.

For example, cysteinyl residues most commonly are reacted withα-haloacetates (and corresponding amides), such as chloroacetic acid orchloroacetamide, to give carboxymethyl or carboxyamidomethylderivatives. Cysteinyl residues also are reacted withbromotrifluoroacetone, α-bromo-β-(4-imidozoyl)propionic acid,chloroacetyl phosphate, N-alkylmaleimides, 3-nitro-2-pyridyl disulfide,methyl 2-pyridyl disulfide, p-chloromercuribenzoate,2-chloromercuri-4-nitrophenol, or chloro-7-nitrobenzo-2-oxa-1,3-diazole.Histidyl residues are reacted with diethylpyrocarbonate at pH 5.5-7.0because this agent is relatively specific for the histidyl side chain.Para-bromophenacyl bromide also is useful; the reaction is generallyperformed in 0.1 M sodium cacodylate at pH 6.0. Lysinyl and aminoterminal residues are reacted with succinic acid or other carboxylicacid anhydrides. Other suitable reagents for reacting withα-amino-containing residues include imidoesters such as methylpicolinimidate; pyridoxal phosphate; pyridoxal; chloroborohydride;trinitrobenzenesulfonic acid; O-methylisourea; and 2,4-pentanedione.Arginyl residues are reacted with one or several conventional reagents,among them phenylglyoxal, 2,3-butanedione, or 1,2-cyclohexanedione.Carboxyl side groups (aspartyl or glutamyl) are reacted withcarbodiimides (R—N═C═N—R′), where R and R′ are different alkyl groups,such as 1-cyclohexyl-3-(2-morpholinyl-4-ethyl)carbodiimide or1-ethyl-3-(4-azonia-4,4-dimethylpentyl)carbodiimide.

The binding moiety of the linker domain will in some embodiments includean alkylating agent, acylating agent, ketone, aldehyde, sulphonate or aphosphorylating agent. Examples of particular binding moieties include,but are not limited to fluorophosphonyl, fluorophosphoryl,fluorosulfonyl, alpha-haloketones or aldehydes or their ketals oracetals, respectively, alpha-haloacyls, nitriles, sulfonated alkyl oraryl thiols, iodoacetylamide group, maleimides, sulfonyl halides andesters, isocyanates, isothiocyanantes, tetrafluorophenyl esters,N-hydroxysuccinimidyl esters, acid halides, acid anhydrides, unsaturatedcarbonyls, alkynes, hydroxamates, alpha-halomethylhydroxamates,aziridines, epoxides, or arsenates and their oxides. Suitable sulfonylgroups include sulfonates, sulfates, sulfinates, sulfamates, etc., ineffect, any reactive functionality having a sulfur group bonded to twooxygen atoms. Suitable epoxides include aliphatic, aralkyl,cycloaliphatic and spiro epoxides.

Non-Covalent Stable Association

In some embodiments, the stable association between the syntheticregulator and the polypeptide is a non-covalent association.Non-covalent associations include, e.g., biotin-avidin linkages, wherethe polypeptide comprises a biotin moiety and the synthetic linkerincludes a linker domain comprising an avidin moiety. Non-covalentassociations further include metal ion-binding associations; andarsenic-binding associations. For example, in some embodiments, theligand-binding polypeptide comprises two or more histidine residuespositioned such that they bind a metal such as Ni⁺², Co⁺², Fe⁺³, Al⁺³,Zn⁺² or Cu⁺²; and the linker domain of the synthetic regulator includesa binding moiety that coordinates the metal ion. For metal-chelatingamino acid sequences, see, e.g., U.S. Pat. No. 5,284,933; U.S. Pat. No.5,310,663; U.S. Pat. No. 4,569,794; U.S. Pat. No. 5,594,115; and U.S.Patent Publication No. US2002/0164718. Metal-chelating amino acidsequences include, but are not limited to, (His)₆. As anothernon-limiting example, in some embodiments the ligand-binding polypeptidecomprises a tetra-cysteine motif; and the linker domain of the syntheticregulator includes a binding moiety that contains arsenic. See, e.g.,U.S. Pat. No. 6,831,160.

Other means of forming a stable association between a syntheticregulator and a ligand-binding polypeptide include tyrosine andtryptophan derivatization chemistry; click chemistry; benzyl guaninetransferase; and the like.

The binding moiety of the linker domain will in some embodiments includea functional group commonly used in photoaffinity labeling, such asbenzophenones, aryl azides, diazonium ions, diazo compounds, anddiazirines. See e.g. Hermanson (1996) Bioconjugate Techniques, AcademicPress.

Ligand

As used herein, the term “ligand” refers to a molecule (e.g., a smallmolecule, a peptide, or a protein) that binds to a polypeptide andeffects a change in an activity of the polypeptide, and/or effects achange in conformation of the polypeptide, and/or affects binding ofanother polypeptide to the polypeptide. Ligands include agonists,partial agonists, inverse agonists, antagonists, allosteric modulators,and blockers.

In some embodiments, the ligand is a naturally-occurring ligand. Inother embodiments, the ligand is a synthetic ligand. In otherembodiments, the ligand is an endogenous ligand. In some embodiments,the ligand is an agonist. In other embodiments, the ligand is an inverseagonist. In other embodiments, the ligand is a partial agonist. In otherembodiments, the ligand is an antagonist. In other embodiments, theligand is an allosteric modulator. In other embodiments, the ligand is ablocker. The term “antagonist” generally refers to an agent that bindsto a ligand-binding polypeptide and inhibits an activity of theligand-binding polypeptide. An “antagonist” may be an agent that bindsto an allosteric site but does not activate the ligand-bindingpolypeptide; instead, the antagonist generally excludes binding by anagonist and thus prevents or hinders activation. The term “blocker”refers to an agent that acts directly on the active site, pore, orallosteric site. Ligands suitable for use herein bind reversibly to aligand-binding site of a ligand-binding polypeptide.

The ligand is selected based in part on the activity of the polypeptideto which the synthetic regulator will be attached. For example, a ligandfor a hormone-binding transcription factor is a hormone, or a syntheticanalog of the hormone. A ligand for a tetracycline transactivator istetracycline or a synthetic analog thereof. A ligand for an enzyme willin some embodiments be a synthetic agonist or antagonist of the enzyme.In some embodiments, a ligand will block the ligand-binding site. Aligand for a ligand-gated ion channel will in some embodiments be anaturally-occurring ligand, or a synthetic version of the ligand, e.g.,a synthetic analog of the ligand. In some embodiments, the ligand isother than an acetylcholine receptor ligand. In some embodiments, theligand is other than trimethylammonium.

In some embodiments, a ligand is a small molecule ligand. Small moleculeligands generally have a molecular weight in a range of from about 50daltons to about 3000 daltons, e.g., from about 50 daltons to about 75daltons, from about 75 daltons to about 100 daltons, from about 100daltons to about 250 daltons, from about 250 daltons to about 500daltons, from about 500 daltons to about 750 daltons, from about 750daltons to about 1000 daltons, from about 1000 daltons to about 1250daltons, from about 1250 daltons to about 1500 daltons, from about 1500daltons to about 2000 daltons, from about 2000 daltons to about 2500daltons, or from about 2500 daltons to about 3000 daltons.

In other embodiments, a ligand is a peptide ligand. Peptide ligands canhave a molecular weight in a range of from about 1 kDa to about 20 kDa,e.g., from about 1 kDa to about 2 kDa, from about 2 kDa to about 5 kDa,from about 5 kDa to about 7 kDa, from about 7 kDa to about 10 kDa, fromabout 10 kDa to about 12 kDa, from about 12 kDa to about 15 kDa, or fromabout 15 kDa to about 20 kDa.

Suitable ligands include, but are not limited to, ligands that block oractivate the function of a ligand-binding protein, where ligand-bindingproteins include channels; receptors (including, but not limited to,ionotropic receptors that bind transmitters; ionotropic receptors thatbind hormones; metabotropic receptors; receptor tyrosine kinases; growthfactor receptors; and other membrane receptors that signal by binding tosoluble or membrane-bound or extracellular matrix-bound small moleculesor proteins); transporters (including but not limited to iontransporters, organic molecule transporters, peptide transporters, andprotein transporters); enzymes (including but not limited to kinases;phosphatases; ubiquitin ligases; acetylases; oxo-reductases; lipases;enzymes that add lipid moieties to proteins or remove them; proteases;and enzymes that modify nucleic acids, including but not limited toligases, helicases, topoisomerases, and telomerases); motor proteins(including kinesins, dyenins and other microtobule-based motors, myosinsand other actin-based motors, DNA and RNA polymerases and other motorsthat track along polynucleotides); scaffolding proteins; adaptorproteins; cytoskeletal proteins; and other proteins that localize ororganize protein domains and superstructures within cells.

Suitable ligands include, but are not limited to, ligands that functionas general anesthetics; ligands that function as local anesthetics;ligands that function as analgesics; synthetic and semi-synthetic opioidanalgesics (e.g., phenanthrenes, phenylheptylamines, phenylpiperidines,morphinans, and benzomorphans) where exemplary opioid analgesics includemorphine, oxycodone, fentanyl, pentazocine, hydromorphone, meperidine,methadone, levorphanol, oxymorphone, levallorphan, codeine,dihydrocodeine, hydrocodone, propoxyphene, nalmefene, nalorphine,naloxone, naltrexone, buprenorphine, butorphanol, nalbuphine, andpentazocine; ionotropic glutamate receptor agonists and antagonists,e.g., N-methyl-D-aspartate (NMDA) receptor agonists and antagonists,kainate (KA) receptor agonists and antagonists, andα-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA) receptoragonists and antagonists; non-opioid analgesics, e.g., acetylsalicylicacid, choline magnesium trisalicylate, acetaminophen, ibuprofen,fenoprofen, diflusinal, and naproxen; muscarinic receptor agonists;muscarinic receptor antagonists; acetylcholine receptor agonists;acetylcholine receptor antagonists; serotonin receptor agonists;serotonin receptor antagonists; enzyme inhibitors; a benzodiazepine,e.g. chlordiazepoxide, clorazepate, diazepam, flurazepam, lorazepam,oxazepam, temazepam or triazolam; a barbiturate sedative, e.g.amobarbital, aprobarbital, butabarbital, butabital, mephobarbital,metharbital, methohexital, pentobarbital, phenobartital, secobarbital,talbutal, theamylal, or thiopental; an H₁ antagonist having a sedativeaction, e.g. diphenhydramine, pyrilamine, promethazine,chlorpheniramine, or chlorcyclizine; an NMDA receptor antagonist, e.g.dextromethorphan ((+)-3-hydroxy-N-methylmorphinan) or its metabolitedextrorphan ((+)-3-hydroxy-N-methylmorphinan), ketamine, memantine,pyrroloquinoline quinine, cis-4-(phosphonomethyl)-2-piperidinecarboxylicacid, budipine, topiramate, neramexane, or perzinfotel; analpha-adrenergic, e.g. doxazosin, tamsulosin, clonidine, guanfacine,dexmetatomidine, modafinil, phentolamine, terazasin, prazasin or4-amino-6,7-dimethoxy-2-(5-methane-sulfonamido-1,2,3,4-tetrahydroisoquino-1-2-yl)-5-(2-pyridyl)quinazoline; a tricyclic antidepressant, e.g. desipramine, imipramine,amitriptyline, or nortriptyline; an anticonvulsant, e.g. carbamazepine,lamotrigine, topiratmate, or valproate; a tachykinin (NK) antagonist,particularly an NK-3, NK-2 or NK-1 antagonist, e.g.(αR,9R)-7-[3,5-bis(trifluoromethyl)benzyl]-8,9,10,11-tetrahydro-9-m-ethyl-5-(4-methylphenyl)-7H-[1,4]diazocino[2,1-g][1,7]-naphthyridine-6-13-dione(TAK-637),5-[[(2R,3S)-2-[(1R)-1-[3,5-bis(trifluoromethyl)phenyl]ethoxy-3-(4-fluorophenyl)-4-morpholinyl]-methyl]-1,2-dihydro-3H-1,2,4-triazol-3-one(MK-869), aprepitant, lanepitant, dapitant or3-[[2-methoxy-5-(trifluoromethoxy)phenyl]-methylamino]-2-phenylpiperidine(2S,3S); a muscarinic antagonist, e.g oxybutynin, tolterodine,propiverine, tropsium chloride, darifenacin, solifenacin, temiverine, oripratropium; a cyclooxygenase-2 (COX-2) selective inhibitor, e.g.celecoxib, rofecoxib, parecoxib, valdecoxib, deracoxib, etoricoxib, orlumiracoxib; a vanilloid receptor agonist (e.g. resinferatoxin) orantagonist (e.g. capsazepine); a beta-adrenergic such as propranolol; a5-HT receptor agonist or antagonist, e.g., a 5-HT_(1B/1D) agonist suchas eletriptan, sumatriptan, naratriptan, zolmitriptan or rizatriptan; a5-HT_(2A) receptor antagonist such asR(+)-α-(2,3-dimethoxy-phenyl)-1-[2-(4-fluorophenylethyl)]-4-piperidin-emethanol(MDL-100907); and the like.

Other suitable ligands include, but are not limited to, lidocaine,novocaine, xylocalne, lignocaine, novocaine, carbocaine, etidocaine,procaine, prontocaine, prilocalne, bupivacaine, cinchocaine,mepivacaine, quinidine, flecamide, procaine,N-[[2′-(aminosulfonyl)biphenyl-4-yl]methyl]-N′-(2,2′-bithien-5-ylmethyl)succinamide(BPBTS), QX-314, saxitoxin, tetrodotoxin, and a type III conotoxin.

Suitable ligands for K⁺ channels include, but are not limited to,quaternary ammonium (e.g., tetraethyl ammonium, tetrabutylammonium,tetrapentylammonium), 4-aminopyridine, sulfonylurea, Glibenclamide;Tolbutamide; Phentolamine, qiunine, qunidine, peptide toxins (e.g.,charybdotoxin, agitoxin-2, apamin, dendrotoxin, VSTX1, hanatoxin-1,hanatoxin-2, and Tityus toxin K-α. Suitable ligands for Na⁺ channelsinclude, but are not limited to, tetrodotoxin, saxitoxin, guanidinium,polyamines (e.g. spermine, cadaverine, putrescine, μ-conotoxin, andδ-conotoxin.

Suitable ligands for cyclic nucleotide gated (CNG) andhyperpolarization-activated cyclic nucleotide-modulated (HCN) channelsinclude, but are not limited to, 1-cis diltiazem and ZD7288. Suitableligands for glycine receptors include, but are not limited to,strychnine and picrotoxin. Suitable ligands for nicotinic acetylcholinereceptors include, but are not limited to, (+)tubocurarine,Methyllycaconitine, gallamine, Nicotine; Anatoxin A, epibatidine,ABT-94, Lophotoxin, Cytisine, Hexamethonium, Mecamylamine, and Dihydro-βerythroidine.

Suitable ligands for GABA receptors include, but are not limited to,Muscimol, THIP, Procabide, bicuculine, picrotoxin, gabazine, gabapentin,diazepam, clonazepam, flumazenil, a β-carboline carboxylate ethyl ester,baclofen, faclofen, and a barbiturate.

Many suitable ligands will be known to those skilled in the art; and thechoice of ligand will depend, in part, on the target (e.g., receptor,ion channel, enzyme, etc.) to which the ligand binds.

Exemplary Synthetic Regulators

Non-limiting examples of synthetic regulators are depicted below. Thoseskilled in the art will appreciate that changes can be made to theexemplary synthetic regulators, e.g., changes in the length, stiffness,charge distribution, or bend angle of the photoisomerizable group,and/or changes in the linker moiety, and/or changes in the ligand can bemade, according to the particular use of the synthetic regulator,according to the protein to which the synthetic regulator is targeted,etc.

Exemplary synthetic regulator 1 comprises a maleimide moiety forattachment to a cysteine residue in a protein; an azobenzene as aphotoisomerizable moiety; and a quaternary ammonium group as the ligand(e.g., a ligand that blocks an ion channel). This synthetic regulator isreferred to as MAL-AZO-QA in Example 1, and has the following structure:

Exemplary synthetic regulator 2 is referred to as MAG-1 in Example 2.Exemplary synthetic regulator-2 comprises a maleimide moiety forattachment to a cysteine residue in a protein; an azobenzene as aphotoisomerizable moiety; and a glutamate moiety as ligand agonist.Exemplary synthetic regulator 2 has the following structure:

Exemplary synthetic regulator 3 is referred to as MAG-2 in Example 3.Exemplary synthetic regulator 3 comprises a maleimide moiety forattachment to a cysteine residue in a protein; an azobenzene as aphotoisomerizable moiety; and a glutamate analog as the ligand moiety.MAG-2 differs from MAG-1 in that the photoisomerizable group in MAG-2 islonger than the photoisomerizable group in MAG-1. Exemplary syntheticregulator 3 has the following structure:

Exemplary synthetic regulator 4 is referred to as AAQ in Example 4.Exemplary synthetic regulator 4 comprises the reactive electrophileacrylamide for attachment to an amino acid residue in a target protein;an azobenzene as a photoisomerizable moiety; and a quaternary ammoniumgroup as the ligand. Exemplary synthetic regulator 4 has the followingstructure:

Exemplary synthetic regulator 5 is referred to as CAQ in Example 4.Exemplary synthetic regulator 5 comprises the reactive electrophilechloroacetamide for attachment to an amino acid residue in a targetprotein; an azobenzene as a photoisomerizable moiety; and a quaternaryammonium group as the ligand. Exemplary synthetic regulator 5 has thefollowing structure:

Compositions

The present invention further provides compositions comprising asynthetic regulator. Compositions comprising a subject syntheticregulator will in many embodiments include one or more of: a salt, e.g.,NaCl, MgCl, KCl, MgSO₄, etc.; a buffering agent, e.g., a Tris buffer,N-(2-Hydroxyethyl)piperazine-N′-(2-ethanesulfonic acid) (HEPES),2-(N-Morpholino)ethanesulfonic acid (MES),2-(N-Morpholino)ethanesulfonic acid sodium salt (MES),3-(N-Morpholino)propanesulfonic acid (MOPS),N-tris[Hydroxymethyl]methyl-3-aminopropanesulfonic acid (TAPS), etc.; asolubilizing agent; a detergent, e.g., a non-ionic detergent such asTween-20, Nonidet-P40, etc.; a protease inhibitor; and the like. In someembodiments, a subject composition comprising a subject syntheticregulator is a pharmaceutical composition, as described in more detailbelow.

Light-Regulated Polypeptides

The present invention further provides a light-regulated polypeptide,where a subject light-regulated polypeptide comprises a ligand-bindingpolypeptide and a subject synthetic regulator of protein function instable association with the ligand-binding polypeptide. The syntheticregulator of protein function is stably associated with theligand-binding polypeptide at or near a ligand binding site of theligand-binding polypeptide. In some embodiments, a subjectlight-regulated polypeptide is isolated, e.g., free of otherpolypeptides or other macromolecules. In other embodiments, a subjectlight-regulated polypeptide is membrane-associated and is present invitro. In other embodiments, a subject light-regulated polypeptide ispresent in a living cell in vitro or in vivo. In other embodiments, asubject light-regulated polypeptide is present in a membrane of a livingcell in vitro or in vivo. In other embodiments, a subjectlight-regulated polypeptide is present in a living cell in a tissue invitro or in vivo. In other embodiments, a subject light-regulatedpolypeptide is present in a living cell in a multicellular organism.

The synthetic regulator is stably associated with the ligand-bindingpolypeptide or near a ligand binding site of a ligand-bindingpolypeptide. For example, the synthetic regulator is stably associatedwith an amino acid side chain or other linkage group (e.g., a sugarmoiety, a high affinity moiety such as biotin, etc.) in theligand-binding polypeptide such that, in one configuration, the ligandbinds to the ligand binding site and effects a change in an activity ofthe polypeptide. In some embodiments, the synthetic regulator is stablyassociated with a linkage group that is from about 1 Å to about 50 Åaway from the ligand binding site, e.g., the synthetic regulator iscovalently linked to a site that is from about 1 Å to about 5 Å, fromabout 5 Å to about 7 Å, from about 7 Å to about 10 Å, from about 10 Å toabout 15 Å, from about 15 Å to about 20 Å, from about 20 Å to about 25Å, from about 25 Å to about 30 Å, from about 30 Å to about 35 Å, fromabout 35 Å to about 40 Å, from about 40 Å to about 45 Å, or from about45 Å to about 50 Å from the ligand binding site.

A change in the wavelength and/or intensity of light (Δλ) to which thelight-regulated polypeptide is exposed results in a change in ligandbinding to a ligand-binding site of the light-regulated polypeptide,e.g., results in a change in binding of the ligand portion of thesynthetic regulated to the ligand-binding site of the light-regulatedpolypeptide. A “change in the wavelength of light to which thelight-regulated polypeptide is exposed” includes: 1) a change from λ₁ toλ₂; 2) a change from λ₂ to λ₁; 3) a change from λ₁ to darkness (nolight); and 4) a change from darkness to λ₁. Repetitive changing from λ₁to λ₂, then from λ₂ to λ₁, and back, e.g., switching from a firstwavelength to a second wavelength, and back again repeatedly, is alsocontemplated. Repetitive changing from light to darkness, from darknessto light, etc., is also contemplated.

In some embodiments, the change in wavelength (from λ₁ to λ₂; from lightto darkness; or from darkness to light) results in a change in bindingof the ligand to a ligand-binding site. As used herein, a “change inbinding of a ligand to a ligand-binding site” includes increased bindingand decreased binding. As used herein, “increased binding” includes oneor more of: an increased probability of binding of the ligand to theligand-binding site; an increased binding affinity of the ligand for theligand-binding site; an increased local concentration of the ligand atthe ligand-binding site; and an increased occupancy of the ligand in theligand-binding site. As used herein, “decreased binding” includes one ormore of: a decreased probability of binding of the ligand to theligand-binding site; a decreased binding affinity of the ligand for theligand-binding site; a decreased local concentration of the ligand atthe ligand-binding site; and a decreased occupancy of the ligand in theligand-binding site. As used herein, the term “change in wavelength” towhich a synthetic regulator is exposed, or to which a ligand-bindingpolypeptide/synthetic light regulator complex is exposed, refers to achange in wavelength from λ₁ to λ₂; a change from light to darkness; ora change from darkness to light. An increase in binding includes anincrease of from about 10% to about 50%, from about 50% to about 2-fold,from about 2-fold to about 5-fold, from about 5-fold to about 10-fold,from about 10-fold to about 50-fold, from about 50-fold to about10²-fold, from about 10²-fold to about 10⁴-fold, from about 10⁴-fold toabout 10⁶-fold, from about 10⁶-fold to about 10⁸-fold, or a greater than10⁸-fold increase in binding. A decrease in binding includes a decreaseof from about 5% to about 10% to about 20% to about 30%, from about 30%to about 40%, from about 40% to about 50%, from about 50% to about 60%,from about 60% to about 70%, from about 70% to about 80%, from about 80%to about 90%, or from about 90% to 100% decrease in binding.

For example, in some embodiments, the ligand has a first probability ofbinding to the ligand site at a first wavelength of light; the ligandhas a second probability of binding to the ligand binding site at asecond wavelength of light; and the second probability is lower than thefirst probability. In other embodiments, the ligand has a firstprobability of binding to the ligand site at a first wavelength oflight; the ligand has a second probability of binding to the ligandbinding site at a second wavelength of light; and the second probabilityis higher than the first probability. In other embodiments, ligand has afirst probability of binding to the ligand site when exposed to light;the ligand has a second probability of binding to the ligand bindingsite in the absence of light (e.g., in darkness); and the secondprobability is lower than the first probability. In other embodiments,the ligand has a first probability of binding to the ligand site whenexposed to light; the ligand has a second probability of binding to theligand binding site in the absence of light and the second probabilityis higher than the first probability.

The local concentration of the ligand portion of the synthetic regulatorat the ligand binding site in a subject light-regulated polypeptide ishigh. For example, the local concentration of the ligand portion of thesynthetic regulator at the ligand binding site in a subjectlight-regulated polypeptide ranges from about 500 nM to about 50 mM,e.g., from about 500 nM to about 750 nM, from about 750 nM to about 1mM, from about 1 mM to about 5 mM, from about 5 mM to about 10 mM, fromabout 10 mM to about 20 mM, from about 20 mM to about 30 mM, or fromabout 30 mM to about 50 mM.

Change in Wavelength Resulting in Binding of the Ligand to theLigand-Binding Site or Higher Affinity Ligand Binding to Ligand-BindingSite

In some embodiments, a change in the wavelength of light to which thelight-regulated polypeptide is exposed results in an increase in bindingaffinity of the ligand portion of a subject synthetic regulator for aligand-binding site of the light-regulated polypeptide. For example, insome embodiments, a change in wavelength of light to which thelight-regulated polypeptide is exposed results in an at least about 10%,at least about 20%, at least about 30%, at least about 50%, at leastabout 75%, at least about 2-fold, at least about 5-fold, at least about10-fold, at least about 25-fold, at least about 50-fold, at least about100-fold, at least about 250-fold, at least about 500-fold, at leastabout 10³-fold, at least about 5×10³-fold, at least about 10⁴-fold, atleast about 5×10⁴-fold, or greater, increase in binding affinity.

Where the ligand is an agonist, the change in wavelength will in someembodiments result in activation of the light-regulated polypeptide.Where the ligand is an agonist, the change in wavelength will in someembodiments result in desensitization of the light-regulatedpolypeptide. Conversely, where the ligand is an antagonist, the changein wavelength results in a block of activation of the light-regulatedpolypeptide, e.g., block of the ability to activate the light-regulatedpolypeptide with free agonist. Where the ligand is a blocker (e.g., apore blocker of an ion channel, an active site blocker of an enzyme, oran interaction domain that binds to other biological macromolecules suchas polypeptides or nucleic acids), the change in wavelength results inblock of polypeptide activity.

Expressed another way, where the ligand is an agonist, and where achange in the wavelength of light to which the light-regulatedpolypeptide is exposed results in a higher binding affinity of theligand moiety of the synthetic regulator to the ligand-binding site ofthe light-regulated polypeptide, the change in wavelength results intransition from an inactive state to an active state, or to adesensitized state. Where the ligand is an antagonist, the change inwavelength results in transition from a responsive state to anunresponsive state. Where the ligand is a blocker, the change inwavelength results in transition from an active state to an inactivestate.

Change in Wavelength Resulting in Removal of Ligand from Ligand-BindingSite, or Reduced Binding Affinity

In some embodiments, a change in the wavelength of light to which thelight-regulated polypeptide is exposed results in removal of the ligandportion of a subject synthetic regulator from a ligand-binding site ofthe light-regulated polypeptide, e.g., the ligand is not bound to theligand-binding site. In some embodiments, a change in the wavelength oflight to which the light-regulated polypeptide is exposed results inreduced binding affinity of the ligand portion of a subject syntheticregulator for a ligand-binding site of the light-regulated polypeptide,e.g., the ligand has reduced binding affinity for he ligand-bindingsite. For example, in some embodiments, a change in the wavelength oflight to which the light-regulated polypeptide is exposed results in areduction of binding affinity of at least about 10%, at least about 20%,at least about 25%, at least about 30%, at least about 40%, at leastabout 50%, at least about 60%, at least about 70%, at least about 80%,at least about 90%, at least about 95%, or more.

Where the ligand is an agonist, the change in wavelength will in someembodiments result in deactivation of the light-regulated polypeptide.Where the ligand is an agonist, the change in wavelength will in someembodiments result in recovery from desensitization of thelight-regulated polypeptide. Conversely, where the ligand is anantagonist, the change in wavelength results in activation of thelight-regulated polypeptide, or results in removal of a blocker from thelight-regulated polypeptide. Where the ligand is a blocker (e.g., a poreblocker of an ion channel, an active site blocker of an enzyme, or aninteraction domain that binds to other biological macromolecules such aspolypeptides or nucleic acids), the change in wavelength results inrelief of a block in polypeptide activity and permits the polypeptide tofunction normally.

Expressed another way, where the ligand is an agonist, and where achange in the wavelength of light to which the light-regulatedpolypeptide is exposed results in removal (or non-binding) of the ligandmoiety of the synthetic regulator from the ligand-binding site of thelight-regulated polypeptide, the change in wavelength results intransition from an active state to an inactive state, or from adesensitized state to a responsive state. Where the ligand is anantagonist, the change in wavelength results in transition from anunresponsive state to a responsive state. Where the ligand is a blocker,the change in wavelength results in transition from an inactive state toan active state.

In some embodiments, the polypeptide is an enzyme, and the ligandbinding site is a catalytic active site. In other embodiments, theligand binding site is an allosteric site of the polypeptide. In otherembodiments, the ligand binding site is a pore of an ion channel. Inother embodiments, the ligand binding site is an interaction motif ordomain through which the polypeptide interacts with other molecules(e.g., polypeptides; nucleic acids).

Ligand-Binding Polypeptides

Suitable ligand-binding polypeptides include any polypeptide having aligand binding site. Suitable polypeptides include, but are not limitedto, enzymes; ion channels; transporters; receptors; motor proteins;scaffolding proteins; adaptors; membrane trafficking proteins;cytoskeleton proteins; and transcription factors. Exemplary,non-limiting ligand-binding polypeptides include ligand-gated ionchannels, receptor tyrosine kinases, G-protein coupled receptors, ionpumps; amino acid transporters; proteins involved in secretion; and thelike. In some embodiments, the ligand-binding polypeptide is other thanan acetylcholine receptor.

Enzymes include, but are not limited to, lipases; synthases; epoxidases;phosphorylases; kinases; oxidoreductases, e.g., oxidases,dehydrogenases, reductases, peroxidases, hydroxylases, and oxygenases;acylases; hydrolases, e.g., esterases, phosphatases, glycosidases,proteases, and peptidases; lyases, e.g., decarboxylases, aldolases, anddehydratases; transferases, e.g., sulfotransferases, aminotransferases,and transpeptidases; isomerases, e.g., racemases, epimerases, cis-transisomerases, intramolecular oxidoreductases, and intramoleculartransferases; ligases, e.g., DNA ligases, amino acid-RNA ligases,acid-thiol ligases, amide synthetases, peptide synthetases, andcyclo-ligases; and the like. In some embodiments, the ligand-bindingprotein is an enzyme chosen from lipases, esterases, proteases,glycosidases, glycosyl transferases, phosphatases, kinases, mono- anddioxygenases, haloperoxidazes, lignin peroxidases, diarylpropaneperoxidazes, epozide hydrolazes, nitrile hydratases, nitrilases,transaminases, amidases, acylases, helicases, topoisomerases,polymerases, and synthetases.

Ion channels include, but are not limited to, cation channels; sodiumion channels; potassium ion channels (where potassium channels include,e.g., a Kv1 potassium channel; a Kv2 potassium channel; a Kv3 potassiumchannel; a Kv4 potassium channel; an HCN potassium channel, e.g., HCN1,HCN2; a HERG potassium channel; an EAG potassium channel; calcium ionchannels; chloride ion channels; cyclic nucleotide-gated channels;2-transmembrane domain channels, including channels selective forpotassium, and less selective cation channels; water and glycerolchannels; proton channels; and the like. In some embodiments, a cationchannel is a voltage-gated cation channel, e.g., a voltage-gated sodiumchannel (e.g., Nav), a voltage-gated calcium channel (e.g., Cav), avoltage-gated potassium channel (e.g., Kv), or a proton channel (Hv). Insome embodiments, an ion channel is an inward rectifier potassiumchannel (e.g., a member of the Kir family). In some embodiments, the ionchannel is a ligand-gated ion channel. A variety of ligand-gated ionchannels are known in the art. Suitable ion channels include pentamericreceptors, e.g., nicotinic acetylcholine receptors; gamma aminobutyricacid (GABA) receptors; glycine receptors; and 5-hydroxytryptamine (5-HT)receptors. Other ligand-gated ion channels include a Ptxr channer, aglycine receptor (GlyR), ASIC, etc. Suitable ion channels also includetetrameric receptors, e.g., glutamate receptors, includingN-methyl-D-aspartate (NMDA) receptors, non-NMDA receptors,α-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA) receptors,an inotropic glutamate receptor (e.g., iGluR6), and kainate (KA)receptors; purinergic receptors, e.g., P1 receptors, P2 receptors; andthe like. For a description of various potassium channels, see, e.g.,Doyle et al. ((1998) Science 280:69-77) and references cited therein. Insome embodiments, an ion channel is a G protein-coupled receptor (GPCR),e.g., a muscarinic GPCR, a dopaminergic GPCR, a serotonergic GPCR, anadrenergic GPCR, an opiate GPCR, a glutamate GPCR, a cannabanoid GPCR, apeptidergic GPCR, an olfactory GPCR, a gustatory GPCR, etc.

Transcription factors include, but are not limited to, ligand-bindingproteins that control transcription and that are inducible by theligand. Such proteins include, but are not limited to, an ecdysonereceptor (see, e.g., Koelle et al. Cell 67:59 (1991); Christianson andKafatos, Biochem. Biophys. Res. Comm. 193:1318 (1993); Henrich et al.,Nucleic Acids Res. 18:4143 (1990); and U.S. Pat. No. 6,958,236); aretinoic acid receptor; a glucocorticoid receptor (see, e.g., Picard etal. (1988) Cell 54: 1073-1080); a tetracycline-transactivator protein(tTA) or a tet repressor (tetR) protein of the tetracyclinerepressor/activator system (see, e.g., WO 94/29442; WO 96/40892; and WO96/01313). Suitable transcription factors also include proteins thatenhance or repress transcription in a manner regulated by binding toother proteins or small ligands, e.g., CREB, and helix-loop-helixproteins.

Receptors include, but are not limited to, G-protein coupled receptors,e.g., opioid receptors (e.g., 6-opioid receptors, μ-opioid receptors,κ-opioid receptors), peptide hormone receptors, neurotransmitterreceptors, odorant receptors, nicotinic acetylcholine receptors, adopamine receptor, a muscarinic receptor, a serotonin receptor, and thelike.

Also suitable is the ligand-binding domain of any of the aforementionedproteins. For example, in some embodiments, a subject light-regulatedpolypeptide comprises the ligand-binding domain of a ligand-bindingpolypeptide; and a subject synthetic regulator in stable associationwith the ligand-binding domain. In some embodiments, the ligand-bindingpolypeptide comprises the ligand-binding domain of a ligand-bindingprotein, fused to a heterologous protein. In some embodiments, theligand-binding polypeptide is an isolated ligand-binding domain of aligand-binding protein, e.g., lacking any other domains that may bepresent in the native polypeptide, such as regulatory domains,transmembrane domains, and the like.

In some embodiments, the ligand-binding protein is a wild-typepolypeptide, e.g., the polypeptide has a wild-type or natives amino acidsequence, e.g., an amino acid sequence that has not been altered byrecombinant methods. In other embodiments, the ligand-binding protein isa recombinant polypeptide. In some embodiments, the ligand-bindingprotein is a synthetic polypeptide. Recombinant polypeptides includevariant polypeptides that have been engineered such that the amino acidsequence differs from a wild-type or naturally-occurring polypeptide.Variant polypeptides include polypeptides comprising an amino acidsequence that differs from the amino acid sequence of a correspondingwild-type or naturally-occurring polypeptide by one to 15 amino acids,e.g., where the amino acid sequence has been altered to include an aminoacid that provides for attachment to the binding moiety of the linkerdomain of the synthetic regulator.

In some embodiments, the ligand-binding protein comprises one or moreamino acid substitutions and/or insertions and/or deletions compared tothe amino acid sequence of a naturally-occurring polypeptide. In some ofthese embodiments, the ligand-binding protein is a variantligand-binding protein that comprises one or more amino acidsubstitutions, compared to a naturally-occurring protein, such that thevariant ligand-binding protein comprises a moiety for stable associationof synthetic regulator. For example, in some embodiments, an amino acidin a ligand-binding protein is substituted with a cysteine, and thesynthetic regulator is covalently linked to the cysteine residue.

In some embodiments, the ligand-binding protein is a fusion protein,where the fusion protein includes the ligand-binding protein fusedin-frame to a heterologous protein, e.g., a protein other than theligand-binding protein, where the heterologous protein is also referredto as a “fusion partner.” In some embodiments, the fusion partner islinked to the ligand-binding protein at the N-terminus of theligand-binding protein. In other embodiments, the fusion partner islinked at the C-terminus of the ligand-binding protein. In otherembodiments, the fusion partner is internal to the ligand-bindingprotein.

Suitable fusion partners include, but are not limited to, epitope tags;solubilization domains; polypeptides that provide for insertion into abiological membrane; polypeptides that provide for uptake into a cell,e.g., polypeptides that provide for uptake into the cytoplasm or into anintracellular compartment; polypeptides that selectively bind to nativeproteins, including at essential protein interaction interfaces;polypeptides that provide for subcellular localization; polypeptidesthat provide a detectable signal (e.g., fluorescent proteins;chromogenic proteins; enzymes that generate luminescent, fluorescent, orchromogenic products; and the like).

Suitable fusion partners include, but are not limited to, luciferase(e.g., firefly luciferase and derivatives thereof; Renilla luciferaseand derivatives thereof); β-galactosidase; chloramphenicol acetyltransferase; glutathione S transferase; a green fluorescent protein(GFP), including, but not limited to, a GFP derived from Aequoriavictoria or a derivative thereof, a number of which are commerciallyavailable; a GFP from a species such as Renilla reniformis, Renillamulleri, or Ptilosarcus guernyi, as described in, e.g., WO 99/49019 andPeelle et al. (2001) J. Protein Chem. 20:507-519; any of a variety offluorescent and colored proteins from Anthozoan species, as describedin, e.g., Matz et al. (1999) Nature Biotechnol. 17:969-973, U.S. PatentPublication No. 2002/0197676, or U.S. Patent Publication No.2005/0032085; a red fluorescent protein; a yellow fluorescent protein; aLumio™ tag (e.g., a peptide of the sequence Cys-Cys-Xaa-Xaa-Cys-Cys,where Xaa is any amino acid other than cysteine, e.g., where Xaa-Xaa isPro-Gly, which peptide is specifically bound by a fluorescein derivativehaving two As(III) substituents, e.g.,4′,5′-bis(1,3,2-dithioarsolan-2-yl)fluorescein; see, e.g., Griffin etal. (1998) Science 281:269; Griffin et al. (2000) Methods Enzymol.327:565; and Adams et al. (2002) J. Am. Chem. Soc. 124:6063); and thelike.

Compositions

The present invention further provides compositions comprising a subjectlight-regulated polypeptide. Compositions comprising a subjectlight-regulated polypeptide will in many embodiments include one or moreof: a salt, e.g., NaCl, MgCl, KCl, MgSO₄, etc.; a buffering agent, e.g.,a Tris buffer, N-(2-Hydroxyethyl)piperazine-N′-(2-ethanesulfonic acid)(HEPES), 2-(N-Morpholino)ethanesulfonic acid (MES),2-(N-Morpholino)ethanesulfonic acid sodium salt (MES),3-(N-Morpholino)propanesulfonic acid (MOPS),N-tris[Hydroxymethyl]methyl-3-aminopropanesulfonic acid (TAPS), etc.; asolubilizing agent; a detergent, e.g., a non-ionic detergent such asTween-20, etc.; a protease inhibitor; and the like.

Methods of Generating a Light-Regulated Polypeptide

In some embodiments, a light-regulated polypeptide is generated byreacting a ligand-binding polypeptide with a subject synthetic regulatorin a cell-free in vitro reaction. In other embodiments, alight-regulated polypeptide is generated by affinity labeling aligand-binding polypeptide with a subject synthetic regulator, where theligand-binding polypeptide is a wild-type, native, or endogenouspolypeptide. In some embodiments, the ligand-binding polypeptide isassociated with a living cell (in vitro or in vivo), the cell thatcomprises the ligand-binding polypeptide is contacted with the syntheticregulator, where the synthetic regulator binds to the ligand-bindingpolypeptide.

In some embodiments, the amino acid sequence of a polypeptide ismodified to include an attachment amino acid at or near theligand-binding site, where the attachment amino acid provides anattachment site for the binding moiety of the ligand domain of thesynthetic regulator. For example, a single amino acid substitution iscarried out to introduce a cysteine residue into a polypeptide, wherethe introduced cysteine residue provides a site for attachment of asynthetic regulator, e.g., where the linker domain includes a maleimidemoiety.

As noted above, in some embodiments, a light-regulated polypeptide isgenerated by reacting a ligand-binding polypeptide with a subjectsynthetic regulator, where the ligand portion of the synthetic regulatorbinds to a ligand-binding site of the ligand-binding polypeptide, andthe binding moiety of the linker domain is thus favored to form a stableassociation with any of a number of different amino acid residues nearthe ligand binding site. This method is referred to as “affinitylabeling.” This method is suitable for labeling isolated polypeptides invitro; and is also suitable for labeling a polypeptide present in aliving cell, either in vitro or in vivo. For example, this method issuitable for labeling an endogenous polypeptide present in a livingcell, either in vitro or in vivo. Thus, the present invention provides amethod of conferring light regulation on a polypeptide (e.g., arecombinant polypeptide, an endogenous polypeptide, a nativepolypeptide, a wild-type polypeptide), the method generally involvingcontacting a polypeptide comprising a ligand binding site with a subjectsynthetic regulator, where the ligand moiety of the synthetic regulatorbinds to the ligand-binding site of the polypeptide, where the syntheticregulator comprises a linker domain comprising a reactive electrophilicmoiety, and where, after binding of the ligand to the ligand-bindingsite, the reactive electrophilic moiety binds to an amino acid sidechain of the polypeptide, thereby conferring light regulation on thepolypeptide, such that a light-regulated polypeptide is generated.

Cells

The present invention further provides a cell comprising a subjectlight-regulated polypeptide. A subject cell finds use in a variety ofapplications, e.g., screening applications, such as identification ofagents that modulate the activity of a polypeptide; and researchapplications such as examination of a metabolic pathway, or otherphysiological event. Where the cell is used in a screening assay, thecell can be referred to as a “test cell.”

In some embodiments, the cell is a eukaryotic cell in in vitro cellculture, and is grown as an adherent monolayer, or in suspension. Inother embodiments, the cell is a eukaryotic cell and is part of a tissueor organ, either in vivo or in vitro. In other embodiments, the cell isa eukaryotic cell and is part of a living multicellular organism, e.g.,a protozoan, an amphibian, a reptile, a plant, an avian organism, amammal, a fungus, an algae, a yeast, a marine microorganism, a marineinvertebrate, an arthropod, an isopod, an insect, an arachnid, etc. Inother embodiments, the cell is a prokaryotic cell.

In other embodiments, the cell is a member of archaea, e.g., anarchaebacterium. Archaebacteria include a methanogen, an extremehalophile, an extreme thermophile, and the like. Suitable archaebacteriainclude, but are not limited to, any member of the groups Crenarchaeota(e.g., Sulfolobus solfataricus, Defulfurococcus mobilis, Pyrodictiumoccultum, Thermofilum pendens, Thermoproteus tenax), Euryarchaeota(e.g., Thermococcus celer, Methanococcus thermolithotrophicus,Methanococcus jannaschii, Methanobacterium thermoautotrophicum,Methanobacterium formicicum, Methanothermus fervidus, Archaeoglobusfulgidus, Thermoplasma acidophilum, Haloferax volcanni, Methanosarcinabarkeri, Methanosaeta concilli, Methanospririllum hungatei,Methanomicrobium mobile), and Korarchaeota.

In some embodiments, the cell is of a particular tissue or cell type.For example, where the organism is a plant, the cell is part of thexylem, the phloem, the cambium layer, leaves, roots, etc. Where theorganism is an animal, the cell will in some embodiments be from aparticular tissue (e.g., lung, liver, heart, kidney, brain, spleen,skin, fetal tissue, etc.), or a particular cell type (e.g., neuronalcells, epithelial cells, endothelial cells, astrocytes, macrophages,glial cells, islet cells, T lymphocytes, B lymphocytes, etc.).

A subject cell is in many embodiments a unicellular organism, or isgrown in culture as a single cell suspension, or as monolayer. In someembodiments, a subject cell is a eukaryotic cell. Suitable eukaryoticcells include, but are not limited to, yeast cells, insect cells, plantcells, fungal cells, mammalian cells, and algal cells. Suitableeukaryotic host cells include, but are not limited to, Pichia pastoris,Pichia finlandica, Pichia trehalophila, Pichia koclamae, Pichiamembranaefaciens, Pichia opuntiae, Pichia thermotolerans, Pichiasalictaria, Pichia guercuum, Pichia pijperi, Pichia stiptis, Pichiamethanolica, Pichia sp., Saccharomyces cerevisiae, Saccharomyces sp.,Hansenula polymorphs, Kluyveromyces sp., Kluyveromyces lactis, Candidaalbicans, Aspergillus nidulans, Aspergillus niger, Aspergillus oryzae,Trichoderma reesei, Chrysosporium lucknowense, Fusarium sp., Fusariumgramineum, Fusarium venenatum, Neurospora crassa, Chlamydomonasreinhardtii, and the like.

Suitable mammalian cells include primary cells and immortalized celllines. Suitable mammalian cell lines include human cell lines, non-humanprimate cell lines, rodent (e.g., mouse, rat) cell lines, and the like.Suitable mammalian cell lines include, but are not limited to, HeLacells (e.g., American Type Culture Collection (ATCC) No. CCL-2), CHOcells (e.g., ATCC Nos. CRL9618, CCL61, CRL9096), 293 cells (e.g., ATCCNo. CRL-1573), Vero cells, NIH 3T3 cells (e.g., ATCC No. CRL-1658),Huh-7 cells, BHK cells (e.g., ATCC No. CCL10), PC12 cells (ATCC No.CRL1721), COS cells, COS-7 cells (ATCC No. CRL1651), RAT1 cells, mouse Lcells (ATCC No. CCLI.3), human embryonic kidney (HEK) cells (ATCC No.CRL1573), HLHepG2 cells, and the like.

In some embodiments, the cell is a neuronal cell or a neuronal-likecell. The cells can be of human, non-human primate, mouse, or ratorigin, or derived from a mammal other than a human, non-human primate,rat, or mouse. In some embodiments, the neuronal cell is a primary cellisolated from an animal. In some embodiments, the neuronal cell orneuronal-liked cell is an immortalized cell line. Suitable cell linesinclude, but are not limited to, a human glioma cell line, e.g., SVGp12(ATCC CRL-8621), CCF-STTG1 (ATCC CRL-1718), SW 1088 (ATCC HTB-12), SW1783 (ATCC HTB-13), LLN-18 (ATCC CRL-2610), LNZTA3WT4 (ATCC CRL-11543),LNZTA3WT11 (ATCC CRL-11544), U-138 MG (ATCC HTB-16), U-87 MG (ATCCHTB-14), H4 (ATCC HTB-148), and LN-229 (ATCC CRL-2611); a humanmedulloblastoma-derived cell line, e.g., D342 Med (ATCC HTB-187), Daoy(ATCC HTB-186), D283 Med (ATCC HTB-185); a human tumor-derivedneuronal-like cell, e.g., PFSK-1 (ATCC CRL-2060), SK-N-DZ(ATCCCRL-2149), SK-N-AS (ATCC CRL-2137), SK-N-FI (ATCC CRL-2142), IMR-32(ATCC CCL-127), etc.; a mouse neuronal cell line, e.g., BC3H1 (ATCCCRL-1443), EOC1 (ATCC CRL-2467), C8-D30 (ATCC CRL-2534), C8-S (ATCCCRL-2535), Neuro-2a (ATCC CCL-131), NB41A3 (ATCC CCL-147), SW10 (ATCCCRL-2766), NG108-15 (ATCC HB-12317); a rat neuronal cell line, e.g.,PC-12 (ATCC CRL-1721), CTX TNA2 (ATCC CRL-2006), C6 (ATCC CCL-107), F98(ATCC CRL-2397), RG2 (ATCC CRL-2433), B35 (ATCC CRL-2754), R3 (ATCCCRL-2764), SCP (ATCC CRL-1700), OA1 (ATCC CRL-6538).

In other embodiments, the host cell is a plant cell. Plant cells includecells of monocotyledons (“monocots”) and dicotyledons (“dicots”).Guidance with respect to plant tissue culture may be found in, forexample: Plant Cell and Tissue Culture, 1994, Vasil and Thorpe Eds.,Kluwer Academic Publishers; and in: Plant Cell Culture Protocols(Methods in Molecular Biology 111), 1999, Hall Eds, Humana Press.

Suitable prokaryotic cells include bacteria (e.g., Eubacteria) andarchaebacteria. Suitable archaebacteria include a methanogen, an extremehalophile, an extreme thermophile, and the like. Suitable archaebacteriainclude, but are not limited to, any member of the groups Crenarchaeota(e.g., Sulfolobus solfataricus, Defulfurococcus mobilis, Pyrodictiumoccultum, Thermofilum pendens, Thermoproteus tenax), Euryarchaeota(e.g., Thermococcus celer, Methanococcus thermolithotrophicus,Methanococcus jannaschii, Methanobacterium thermoautotrophicum,Methanobacterium formicicum, Methanothermus fervidus, Archaeoglobusfulgidus, Thermoplasma acidophilum, Haloferax volcanni, Methanosarcinabarkeri, Methanosaeta concilli, Methanospririllum hungatei,Methanomicrobium mobile), and Korarchaeota. Suitable eubacteria include,but are not limited to, any member of Hydrogenobacteria, Thermotogales,Green nonsulfphur bacteria, Denococcus Group, Cyanobacteria, Purplebacteria, Planctomyces, Spirochetes, Green Sulphur bacteria, Cytophagas,and Gram positive bacteria (e.g., Mycobacterium sp., Micrococcus sp.,Streptomyces sp., Lactobacillus sp., Helicobacterium sp., Clostridiumsp., Mycoplasma sp., Bacillus sp., etc.).

Suitable prokaryotic cells include, but are not limited to, any of avariety of laboratory strains of Escherichia coli, Lactobacillus sp.,Salmonella sp., Shigella sp., and the like. See, e.g., Carrier et al.(1992) J. Immunol. 148:1176-1181; U.S. Pat. No. 6,447,784; and Sizemoreet al. (1995) Science 270:299-302. Examples of Salmonella strains whichcan be employed in the present invention include, but are not limitedto, Salmonella typhi and S. typhimurium. Suitable Shigella strainsinclude, but are not limited to, Shigella flexneri, Shigella sonnei, andShigella disenteriae. Typically, the laboratory strain is one that isnon-pathogenic. Non-limiting examples of other suitable bacteriainclude, but are not limited to, Bacillus subtilis, Pseudomonas pudita,Pseudomonas aeruginosa, Pseudomonas mevalonii, Rhodobacter sphaeroides,Rhodobacter capsulatus, Rhodospirillum rubrum, Rhodococcus sp., and thelike. In some embodiments, the cell is Escherichia coli.

Membranes

The present invention further provides a membrane comprising a subjectlight-regulated polypeptide. In some embodiments, the membrane is abiological membrane (e.g., a lipid bilayer surrounding a biologicalcompartment such as a cell, including artificial cells, or a membranevesicle or sheet). In some embodiments, the membrane is part of a livingcell, as described above. In other embodiments, the membrane is anartificial (synthetic) membrane, e.g., a planar membrane, a liposome,etc.

In some embodiments, the artificial membrane is a lipid bilayer. Inother embodiments, the artificial membrane is a lipid monolayer. In someembodiments, the artificial membrane is part of a liposome. Liposomesinclude unilamellar vesicles composed of a single membrane or lipidbilayer, and multilamellar vesicles (MLVs) composed of many concentricmembranes (or lipid bilayers).

Artificial membranes, and methods of making same, have been described inthe art. See, e.g., U.S. Pat. No. 6,861,260; Kansy et al. (1998) J. Med.Chem. 41(7):1007-10; and Yang et al. (1996) Advanced Drug DeliveryReviews 23:229-256.

A subject artificial membrane will in some embodiments, include one ormore phospholipids. In some embodiments, the artificial membranecomprises a mixture of phospholipids containing saturated or unsaturatedmono or disubstituted fatty acids and a combination thereof. Thesephospholipids are in some embodiments selected fromdioleoylphosphatidylcholine, dioleoylphosphatidylserine,dioleoylphosphatidylethanolamine, dioleoylphosphatidylglycerol,dioleoylphosphatidic acid, palmitoyloleoylphosphatidylcholine,palmitoyloleoylphosphatidylserine,palmitoyloleoylphosphatidylethanolamine,palmitoyloleoylphophatidylglycerol, palmitoyloleoylphosphatidic acid,palmitelaidoyloleoylphosphatidylcholine,palmitelaidoyloleoylphosphatidylserine,palmitelaidoyloleoylphosphatidylethanolamine,palmitelaidoyloleoylphosphatidylglycerol,palmitelaidoyloleoylphosphatidic acid,myristoleoyloleoylphosphatidylcholine,myristoleoyloleoylphosphatidylserine,myristoleoyloleoylphosphatidylethanoamine,myristoleoyloleoylphosphatidylglycerol, myristoleoyloleoylphosphatidicacid, dilinoleoylphosphatidylcholine, dilinoleoylphosphatidylserine,dilinoleoylphosphatidylethanolamine, dilinoleoylphosphatidylglycerol,dilinoleoylphosphatidic acid, palmiticlinoleoylphosphatidylcholine,palmiticlinoleoylphosphatidylserine,palmiticlinoleoylphosphatidylethanolamine,palmiticlinoleoylphosphatidylglycerol, and palmiticlinoleoylphosphatidicacid. Suitable phospholipids also include the monoacylated derivativesof phosphatidylcholine (lysophophatidylidylcholine), phosphatidylserine(lysophosphatidylserine), phosphatidylethanolamine(lysophosphatidylethanolamine), phophatidylglycerol(lysophosphatidylglycerol) and phosphatidic acid (lysophosphatidicacid). The monoacyl chain in such lysophosphatidyl derivatives will insome embodiments be palimtoyl, oleoyl, palmitoleoyl, linoleoyl myristoylor myristoleoyl.

Methods of Modulating Protein Activity

The present invention provides methods of modulating protein activity.In certain aspects, the present invention provides methods of modulatingactivity of a subject light-regulated polypeptide, where the methodgenerally involves changing the wavelength of light to which thelight-regulated polypeptide is exposed. In certain aspects, the presentinvention provides methods of modulating activity of a ligand-bindingpolypeptide, where the method generally involves: a) contacting theligand-binding polypeptide with a subject synthetic regulator, where thesynthetic regulator binds to the ligand-binding polypeptide by affinitylabeling, thereby generating a light-regulated polypeptide; and b)changing the wavelength of light to which the light-regulatedpolypeptide is exposed.

As noted above, a “change in the wavelength of light to which thelight-regulated polypeptide is exposed” includes: 1) a change from λ₁ toλ₂; 2) a change from λ₂ to λ₁; 3) a change from λ₁ to darkness (nolight); and 4) a change from darkness to λ₁. In certain aspects, thepresent invention provides methods of modulating activity of a native(wild-type) polypeptide, where the method generally involves: a)contacting a polypeptide with a subject synthetic regulator, where thesubject synthetic regulator binds to the polypeptide, forming asynthetic regulator/polypeptide complex; and b) changing the wavelengthof light to which the synthetic regulator/polypeptide complex isexposed. As noted above, a “change in the wavelength of light to whichthe light-regulated polypeptide is exposed” includes: 1) a change fromλ₁ to λ₂; 2) a change from λ₂ to λ₁; 3) a change from λ₁ to darkness (nolight); and 4) a change from darkness to λ₁. The syntheticregulator/polypeptide complex is also referred to as a “light-regulatedpolypeptide.” In some embodiments, the synthetic regulator/polypeptidecomplex is generated by affinity labeling, as described above.

In some embodiments, the ligand-binding polypeptide or thelight-regulated polypeptide is present in a cell-free in vitro system,e.g, the ligand-binding polypeptide or the light-regulated polypeptideis not associated with a cell. In other embodiments, the ligand-bindingpolypeptide or the light-regulated polypeptide is associated with acell, e.g., the ligand-binding polypeptide or the light-regulatedpolypeptide is integrated into a cell membrane in a cell, theligand-binding polypeptide or the light-regulated polypeptide is in thecytosol of a cell, the ligand-binding polypeptide or the light-regulatedpolypeptide is in an intracellular organelle, etc. In other embodiments,the ligand-binding polypeptide or the light-regulated polypeptide is ina synthetic membrane, e.g., in a planar synthetic membrane, in aliposome, in a membrane of an artificial cell, etc. In some embodiments,the cell-associated ligand-binding polypeptide or the cell-associatedlight-regulated polypeptide is in a cell in vitro, e.g., in a cell in amonolayer, in a cell in suspension, in an in vitro tissue, etc. In otherembodiments, the cell-associated ligand-binding polypeptide or thecell-associated light-regulated polypeptide is in a cell in vivo, e.g.,in a cell of an organism, e.g., a living organism.

In some embodiments, the change in wavelength (from λ₁ to λ₂; from lightto darkness; or from darkness to light) results in a change in bindingof the ligand to a ligand-binding site. As used herein, a “change inbinding of a ligand to a ligand-binding site” includes increased bindingand decreased binding. As used herein, “increased binding” includes oneor more of: an increased probability of binding of the ligand to theligand-binding site; an increased binding affinity of the ligand for theligand-binding site; an increased local concentration of the ligand atthe ligand-binding site; and an increased occupancy of the ligand in theligand-binding site. As used herein, “decreased binding” includes one ormore of: a decreased probability of binding of the ligand to theligand-binding site; a decreased binding affinity of the ligand for theligand-binding site; a decreased local concentration of the ligand atthe ligand-binding site; and a decreased occupancy of the ligand in theligand-binding site. As used herein, the term “change in wavelength” towhich a synthetic regulator is exposed, or to which a ligand-bindingpolypeptide/synthetic light regulator complex is exposed, refers to achange in wavelength from λ₁ to λ₂; a change from light to darkness; ora change from darkness to light. An increase in binding includes anincrease of from about 10% to about 50%, from about 50% to about 2-fold,from about 2-fold to about 5-fold, from about 5-fold to about 10-fold,from about 10-fold to about 50-fold, from about 50-fold to about10²-fold, from about 10²-fold to about 10⁴-fold, from about 10⁴-fold toabout 10⁶-fold, from about 10⁶-fold to about 10⁸-fold, or a greater than10⁸-fold increase in binding. A decrease in binding includes a decreaseof from about 5% to about 10% to about 20% to about 30%, from about 30%to about 40%, from about 40% to about 50%, from about 50% to about 60%,from about 60% to about 70%, from about 70% to about 80%, from about 80%to about 90%, or from about 90% to 100% decrease in binding.

For example, in some embodiments, the ligand has a first probability ofbinding to the ligand site at a first wavelength of light; the ligandhas a second probability of binding to the ligand binding site at asecond wavelength of light; and the second probability is lower than thefirst probability. In other embodiments, the ligand has a firstprobability of binding to the ligand site at a first wavelength oflight; the ligand has a second probability of binding to the ligandbinding site at a second wavelength of light; and the second probabilityis higher than the first probability. In other embodiments, ligand has afirst probability of binding to the ligand site when exposed to light;the ligand has a second probability of binding to the ligand bindingsite in the absence of light (e.g., in darkness); and the secondprobability is lower than the first probability. In other embodiments,the ligand has a first probability of binding to the ligand site whenexposed to light; the ligand has a second probability of binding to theligand binding site in the absence of light and the second probabilityis higher than the first probability.

A change in wavelength can result in a change in activity of thelight-regulated protein. “Activity” will depend, in part, on theligand-binding polypeptide, and can include enzymatic activity (forenzymes); activity of an ion channel; activity of a receptor intransmitting a signal; etc.

In some embodiments, the change in wavelength results in binding of theligand to the ligand-binding site of the light-regulated polypeptide. Insome embodiments, the change in wavelength results in increased bindingaffinity of the ligand to the ligand-binding site for thelight-regulated polypeptide. In these embodiments, where the ligand isan agonist, and the change results in activation of said light-regulatedpolypeptide; and where the ligand is an antagonist, the change resultsin block of activation of the light-regulated polypeptide; and where theligand is an active site or pore blocker, the change results ininhibition of the light-regulated polypeptide; and where the ligand is ablocker of a site of interaction with other macromolecules, the changeinterferes with that interaction. In some embodiments, prolonged bindingof an agonist to the ligand-binding site results in desensitization orinactivation of the light-regulated polypeptide. In other embodiments,binding of an antagonist blocks activation of the light-regulatedpolypeptide.

In other embodiments, the change in wavelength results in lack ofbinding of the ligand to the ligand-binding site, e.g., removal of theligand from the ligand-binding site of the light-regulated polypeptide.In other embodiments, the change in wavelength results in reducedbinding affinity of the ligand for the ligand-binding site, e.g.,reduced binding affinity of ligand for the ligand-binding site of thelight-regulated polypeptide.

In these embodiments, where the ligand is an antagonist, the changeresults in activation of said light-regulated polypeptide; and where theligand is an agonist, the change results in deactivation oflight-regulated polypeptide, or recovery from desensitization orinactivation.

In some embodiments, the light-regulated polypeptide is exposed to lightof a first wavelength, where exposure to light of the first wavelength(λ₁) results in binding of the ligand to the ligand-binding site (orincreased binding affinity of the ligand for the ligand-binding site);and the polypeptide is subsequently exposed to light of a secondwavelength (λ₂), where exposure to light of the second wavelengthresults in removal of the ligand from the ligand-binding site (orreduced binding affinity of the ligand for the ligand-binding site).This change in wavelength from a first wavelength to a second wavelength(λ₂) can be repeated numerous times, such that the light is switchedback and forth between λ₁ and λ₂. Switching between λ₁ and λ₂ results inswitching or transition from a ligand-bound state to a ligand-unboundstate.

In some embodiments, the light-regulated polypeptide is exposed to lightof a first wavelength, where exposure to light of the first wavelength(λ₁) results in binding of the ligand to the ligand-binding site (orincreased binding affinity of the ligand for the ligand-binding site);and the light is subsequently turned off, e.g., the polypeptide is indarkness, where keeping the polypeptide in darkness results in removalof the ligand from the ligand-binding site (or reduced binding affinityof the ligand for the ligand-binding site). This change from λ₁ todarkness can be reversed, e.g., from darkness to λ₁; and repeated anynumber of times, as described above. In other embodiments, a subjectpolypeptide is exposed to light of a first wavelength, where exposure tolight of the first wavelength (λ₁) results in lack of binding of theligand to the ligand-binding site (or reduced binding affinity of theligand for the ligand-binding site); and the light is subsequentlyturned off, e.g., the polypeptide is in darkness, where keeping thepolypeptide in darkness results in binding of the ligand to theligand-binding site (or increased binding affinity of the ligand for theligand-binding site). This change from λ₁ to darkness can be reversed,e.g., from darkness to λ₁; and repeated any number of times, asdescribed above.

As noted above, the change in wavelength can be repeated any number oftimes, e.g, from λ₁ to λ₂ and from λ₂ to λ₁; or from λ₁ to darkness andfrom darkness to λ₁. Thus, a subject method provides for inducing atransition or switch from a ligand-bound state of a protein to aligand-unbound state of the light-regulated protein, or from a highaffinity state to a low affinity state. Depending on whether the ligandis an agonist or an antagonist, the protein will in some embodiments beswitched from an active state to an inactive (or deactivated) state, orfrom an inactive (or deactivated) state to an active state.

The wavelength of light to which the light-regulated polypeptide isexposed ranges from 10⁻⁸ m to about 1 m, e.g., from about 10⁻⁸ m toabout 10⁻⁷ m, from about 10⁻⁷ m to about 10⁻⁶ m, from about 10⁻⁶ m toabout 10⁻⁴ m, from about 10⁻⁴ m to about 10⁻² m, or from about 10⁻² m toabout 1 m. “Light,” as used herein, refers to electromagnetic radiation,including, but not limited to, ultraviolet light, visible light,infrared, and microwave.

The wavelength of light to which the light-regulated polypeptide isexposed ranges in some embodiments from about 200 nm to about 800 nm,e.g., from about 200 nm to about 250 nm, from about 250 nm to about 300nm, from about 300 nm to about 350 nm, from about 350 nm to about 400nm, from about 400 nm to about 450 nm, from about 450 nm to about 500nm, from about 500 nm to about 550 nm, from about 550 nm to about 600nm, from about 600 nm to about 650 nm, from about 650 nm to about 700nm, from about 700 nm to about 750 nm, or from about 750 nm to about 800nm, or greater than 800 nm.

In other embodiments, the wavelength of light to which thelight-regulated polypeptide is exposed ranges from about 800 nm to about2500 nm, e.g., from about 800 nm to about 900 nm, from about 900 nm toabout 1000 nm, from about 1000 nm to about 1200 nm, from about 1200 nmto about 1400 nm, from about 1400 nm to about 1600 nm, from about 1600nm to about 1800 nm, from about 1800 nm to about 2000 nm, from about2000 nm to about 2250 nm, or from about 2250 nm to about 2500 nm. Inother embodiments, the wavelength of light to which the light-regulatedpolypeptide is exposed ranges from about 2 nm to about 200 nm, e.g.,from about 2 nm to about 5 nm, from about 5 nm to about 10 nm, fromabout 10 nm to about 25 nm, from about 25 nm to about 50 nm, from about50 nm to about 75 nm, from about 100 nm, from about 100 nm to about 150nm, or from about 150 nm to about 200 nm.

The difference between the first wavelength and the second wavelengthcan range from about 10 nm to about 800 nm or more, e.g., from about 10nm to about 25 nm, from about 25 nm to about 50 nm, from about 50 nm toabout 100 nm, from about 100 nm to about 200 nm, from about 200 nm toabout 250 nm, from about 250 nm to about 500 nm, or from about 500 nm toabout 800 nm. Of course, where the light-regulated polypeptide isswitched from darkness to light, the difference in wavelength is fromessentially zero to a second wavelength.

The intensity of the light can vary from about 1 W/m² to about 50 W/m²,e.g., from about 1 W/m² to about 5 W/m², from about 5 W/m² to about 10W/m², from about 10 W/m², from about 10 W/m² to about 15 W/m², fromabout 15 W/m² to about 20 W/m², from about 20 W/m² to about 30 W/m²,from about 30 W/m² to about 40 W/m², or from about 40 W/m² to about 50W/m². The intensity of the light can vary from about 1 μW/cm² to about100 μW/cm², e.g., from about 1 μW/cm² to about 5 μW/cm², from about 5μW/cm² to about 10 μW/cm², from about 10 μW/cm² to about 20 μW/cm², fromabout 20 μW/cm² to about 25 μW/cm², from about 25 μW/cm² to about 50μW/cm², from about 50 μW/cm² to about 75 μW/cm², or from about 75 μW/cm²to about 100 μW/cm². In some embodiments, the intensity of light variesfrom about 1 μW/mm² to about 1 W/mm², e.g., from about 1 μW/mm² to about50 μW/mm², from about 50 μW/mm² to about 100 μW/mm², from about 100μW/mm² to about 500 μW/mm², from about 500 μW/mm² to about 1 mW/mm²,from about 1 mW/mm² to about 250 mW/mm², from about 250 mW/mm² to about500 mW/mm², or from about 500 mW/mm² to about 1 W/mm².

In some embodiments, the light-regulated polypeptide is regulated usingsound, instead of electromagnetic (EM) radiation (light). For example,in some embodiments, the light-regulated polypeptide is regulated usingultrasound to effect a change from a first isomeric form to a secondisomeric form.

The duration of exposure of the light-regulated protein to light canvary from about 1 μsecond (μs) to about 60 seconds (s) or more, e.g.,from about 1 μs to about 5 μs, from about 5 μs to about 10 μs, fromabout 10 μs to about 25 μs, from about 25 μs to about 50 μs, from about50 μs to about 100 μs, from about 100 μs to about 250 μs, from about 250μs to about 500 μs, from about 500 μs to about 1 millisecond (ms), fromabout 1 ms to about 10 ms, from about 10 ms to about 50 ms, from about50 ms to about 100 ms, from about 100 ms to about 500 ms, from about 500ms to about 1 second, from about 1 second to about 5 seconds, from about5 seconds to about 10 seconds, from about 10 seconds to about 15seconds, from about 15 seconds to about 30 seconds, from about 30seconds to about 45 seconds, or from about 45 seconds to about 60seconds, or more than 60 seconds. In some embodiments, the duration ofexposure of the light-regulated polypeptide to light varies from about60 seconds to about 10 hours, e.g., from about 60 seconds to about 15minutes, from about 15 minutes to about 30 minutes, from about 30minutes to about 60 minutes, from about 60 minutes to about 1 hour, fromabout 1 hour to about 4 hours, from about 4 hours to about 6 hours, fromabout 6 hours to about 8 hours, or from about 8 hours to about 10 hours,or longer.

The duration of binding of the ligand portion of the synthetic regulatorto the ligand-binding site can vary from less than one second to days.For example, in some embodiments, the duration of binding of the ligandportion of the synthetic regulator to the ligand-binding site variesfrom about 0.5 second to about 1 second, from about 1 second to about 5seconds, from about 5 seconds to about 15 seconds, from about 15 secondsto about 30 seconds, from about 30 seconds to about 60 seconds, fromabout 1 minute to about 5 minutes, from about 5 minutes to about 15minutes, from about 15 minutes to about 30 minutes, or from about 30minutes to about 60 minutes. In other embodiments, the duration ofbinding of the ligand portion of the synthetic regulator to theligand-binding site varies from about 60 minutes to about 2 hours, fromabout 2 hours to about 4 hours, from about 4 hours to about 8 hours,from about 8 hours to about 12 hours, from about 12 hours to about 18hours, from about 18 hours to about 24 hours, from about 24 hours toabout 36 hours, from about 36 hours to about 48 hours, from about 48hours to about 60 hours, from about 60 hours to about 72 hours, fromabout 3 days to about 4 days, from about 4 days to about 5 days, or fromabout 5 days to about 7 days, or longer.

Modulating Activity of a Second, Non-Light-Regulated Polypeptide

In some embodiments, modulating the activity of a light-regulatedpolypeptide results in modulating the activity of a polypeptide otherthan the light-regulated polypeptide. Thus, in other aspects, thepresent invention provides methods of modulating activity of apolypeptide whose activity is modulated by modulating the activity of alight-regulated polypeptide. In some aspects, the present inventionprovides methods of modulating the activity of a non light-regulatedpolypeptide in a cell. The methods generally involve modulating anactivity of a light-regulated polypeptide in the cell, where modulationof the activity of the light-regulated polypeptide in the cell modulatesthe activity of the non-light-regulated polypeptide.

A non-light-regulated polypeptide whose activity is modulated bymodulating the activity of a light-regulated polypeptide includes apolypeptide whose activity is modulated by a change in voltage of abiological membrane, a polypeptide whose activity is modulated bydepolarization of a biological membrane; a polypeptide whose activity ismodulated by a change in intracellular concentration of an ion (e.g., amonovalent or divalent ion, e.g., a monovalent or divalent cation); apolypeptide whose activity is modulated by phosphorylation; and thelike. As one non-limiting example, a light-regulated polypeptidecomprises a glutamate receptor (ligand-gated ion channel) as theligand-binding polypeptide, where the light-regulated polypeptide is inthe plasma membrane of a cell. Light activation of the light-regulatedglutamate receptor in the cell opens the channel, resulting in influx ofion and depolarization of the plasma membrane. Depolarization of theplasma membrane activates a voltage-gated ion channel, such as a calciumchannel. Activation of the calcium channels is readily detected bystandard methods, e.g., use of an indicator dye, etc.). As anothernon-limiting example, the light-regulated polypeptide comprises a GPCRas the ligand-binding polypeptide. Activation of the light-regulatedGPCR activates an ion channel or an enzyme. Activation of the ionchannel or enzyme is readily detected using standard methods, e.g., useof an indicator dye for the permeating ion, or a colorimetric,fluorimetric, or luminescence assay for the product of the enzyme. Asanother non-limiting example, the light-regulated polypeptide comprisesa receptor tyrosine kinase (RTK); and activation of the light-regulatedRTK results in phosphorylation of a downstream protein, e.g., atranscription factor. Activation of the transcription factor is readilydetected by, e.g., detecting a transcript. As another non-limitingexample, the light-regulated polypeptide comprises an opioid receptor.Modulation of the opioid receptor by exposure to light (or removal oflight) can modulate a potassium ion channel; and modulation of apotassium ion channel is readily detected using standard methods, e.g.,use of a dye for potassium ions.

Utility

A subject synthetic regulator, a subject light-regulated polypeptide, asubject cell, and a subject method of modulating protein function, areuseful in a wide variety of research applications, pharmaceuticalapplications, screening assays, therapeutic applications, and the like.

Research Applications

In some embodiments, a subject synthetic regulator or a subjectlight-regulated polypeptide, is useful in studies of cell function, instudies of physiology of whole organisms, and the like. In some aspects,a subject synthetic regulator or a subject light-regulated polypeptidefinds use in controlling gene expression.

For example, a subject synthetic regulator will in some embodimentsinclude a ligand that binds to the ligand-binding site of atranscriptional regulator protein. A transcriptional regulator proteinthat includes such a synthetic regulator in stable association with theprotein will be light regulated, e.g., will be a light-regulatedtranscription factor. Thus, gene expression can be controlled bychanging the wavelength of light to which the light-regulatedtranscription factor is exposed.

In physiological studies, changing light exposure of a tissue, organ, orwhole organism (or a part of a whole organism) that includes alight-regulated protein provides a method of regulating a function inthe tissue, organ, or whole organism. For example, where thelight-regulated protein comprises a ligand-binding protein that is aligand-gated ion channel, and the synthetic regulator comprises theligand for the ligand-gated ion channel, changing the wavelength oflight to which the light-regulated protein is exposed will result inopening or closing of the ion channel, thereby altering ionconcentration in cells in a manner that alters their activity (e.g.,hormone or neurotransmitter secretion) or state (e.g., transcriptionalor translational or metabolic state) or electrical firing, etc.

Screening Methods

The present invention provides methods of identifying an agent thatmodulates a function (e.g., an activity) of a polypeptide. The methodsgenerally involve contacting a light-regulated polypeptide with a testagent; and determining the effect, if any, of the test agent on theactivity of the light-regulated polypeptide (or on the activity of apolypeptide that is regulated by the light-regulated polypeptide). Theeffect, if any, of the test agent on the activity of the light-regulatedpolypeptide is determined by exposing the light-regulated polypeptide tolight of a first wavelength. In the absence of the test agent, exposureof the light-regulated polypeptide to light of a first wavelengthinduces a transition from a ligand-unbound state to a ligand-boundstate. In the presence of a test agent that affects binding of theligand to the ligand-binding site, the transition from theligand-unbound state to a ligand-bound state is inhibited.

In some embodiments, the light-regulated polypeptide is in vitro insolution (e.g., free of cells or membranes); and the assay is carriedout in vitro. In other embodiments, the light-regulated polypeptide isin a membrane (e.g., a synthetic membrane) in the absence of a livingcell (e.g., in a cell-free system); and the assay is carried out invitro. In other embodiments, the light-regulated polypeptide is in acell, e.g., a living cell in vitro or in vivo; and in some embodiments,the assay is carried out in vitro, and in other embodiments, the assayis carried out in vivo.

In some aspects, the present invention provides methods for identifyingan agent that modulates a function (e.g., an activity) of anon-light-regulated polypeptide in the same solution, membrane, or cell,where the activity of the non-light-regulated polypeptide is modulatedby modulating the activity of a light-regulated polypeptide. The methodsgenerally involve contacting a light-regulated polypeptide (where thelight-regulated polypeptide is in a solution, membrane, or cell) with atest agent; and determining the effect, if any, of the test agent on theactivity of the non-light-regulated polypeptide (where the non-lightregulated polypeptide is in the same solution, membrane, or cell as thelight-regulated polypeptide), where the activity of thenon-light-regulated polypeptide is modulated by changing the wavelengthof light to which the cell is exposed. Whether the activity of thenon-light regulated polypeptide is modulated is determined using anassay appropriate to the activity of the non-light-regulatedpolypeptide. For example, where the non-light-regulated polypeptide is acalcium channel, a calcium-sensitive dye, such as a Fura-2 dye, will insome embodiments be used to detect an effect of the test agent on theactivity of the calcium channel. For example, where thenon-light-regulated polypeptide is a sodium channel, a sodium-sensitivedye such as sodium-binding benzofuran isophthalate (SBFI) will in someembodiments be used to detect an effect of the test agent on theactivity of the sodium channel.

In some embodiments, the light-regulated polypeptide is in a cell (e.g.,is integrated into the plasma membrane, is in the cytosol of the cell,is in a subcellular organelle, is in the nucleus of the cell, or isintegrated into a membrane of a subcellular organelle). In theseembodiments, the cell comprising the light-regulated polypeptide is a“test cell.” The methods generally involve contacting the test cell witha test agent; and determining the effect, if any, of the test agent onthe activity of the light-regulated polypeptide.

In some embodiments, the test agent is one that inhibits induction of atransition from a first, ligand-bound state to a second, ligand-unboundstate. For example, in some embodiments, a test agent of interest is onethat inhibits induction of a transition from a first, ligand-unboundstate to a second, ligand-bound state by at least about 5%, at leastabout 10%, at least about 15%, at least about 20%, at least about 25%,at least about 30%, at least about 40%, at least about 50%, at leastabout 60%, at least about 70%, or at least about 80%, or more, comparedto the induction in the absence of the test agent.

The terms “candidate agent,” “test agent,” “agent,” “substance,” and“compound” are used interchangeably herein. Candidate agents encompassnumerous chemical classes, typically synthetic, semi-synthetic, ornaturally-occurring inorganic or organic molecules. Candidate agentsinclude those found in large libraries of synthetic or naturalcompounds. For example, synthetic compound libraries are commerciallyavailable from Maybridge Chemical Co. (Trevillet, Cornwall, UK),ComGenex (South San Francisco, Calif.), and MicroSource (New Milford,Conn.). A rare chemical library is available from Aldrich (Milwaukee,Wis.). Alternatively, libraries of natural compounds in the form ofbacterial, fungal, plant and animal extracts are available from Pan Labs(Bothell, Wash.) or are readily producible.

Candidate agents may be small organic or inorganic compounds having amolecular weight of more than 50 and less than about 2,500 daltons.Candidate agents may comprise functional groups necessary for structuralinteraction with proteins, particularly hydrogen bonding, and mayinclude at least an amine, carbonyl, hydroxyl or carboxyl group, and maycontain at least two of the functional chemical groups. The candidateagents may comprise cyclical carbon or heterocyclic structures and/oraromatic or polyaromatic structures substituted with one or more of theabove functional groups. Candidate agents are also found amongbiomolecules including peptides, saccharides, fatty acids, steroids,purines, pyrimidines, derivatives, structural analogs or combinationsthereof.

Assays of the invention include controls, where suitable controlsinclude a sample (e.g., a sample comprising a subject polypeptide in theabsence of the test agent). Generally a plurality of assay mixtures isrun in parallel with different agent concentrations to obtain adifferential response to the various concentrations. Typically, one ofthese concentrations serves as a negative control, i.e. at zeroconcentration or below the level of detection.

A variety of other reagents may be included in the screening assay.These include reagents like salts, neutral proteins, e.g. albumin,detergents, etc. that are used to facilitate optimal protein-proteinbinding and/or reduce non-specific or background interactions. Reagentsthat improve the efficiency of the assay, such as protease inhibitors,nuclease inhibitors, anti-microbial agents, etc. may be used. Thecomponents of the assay mixture are added in any order that provides forthe requisite binding or other activity. Incubations are performed atany suitable temperature, typically between 4° C. and 40° C. Incubationperiods are selected for optimum activity, but may also be optimized tofacilitate rapid high-throughput screening. Typically between 0.1 hourand 1 hour will be sufficient.

The screening methods may be designed a number of different ways, wherea variety of assay configurations and protocols may be employed, as areknown in the art. The above components of the method may be combined atsubstantially the same time or at different times. In some embodiments,a subject method will include one or more washing steps.

In some embodiments, the ligand-binding, light regulated polypeptide isassayed in a membrane-free, cell free assay. In other embodiments, theligand-binding, light regulated polypeptide is integrated into anartificial membrane. In other embodiments, the ligand-binding, lightregulated polypeptide is integrated into a biological membrane. In otherembodiments, the ligand-binding, light regulated is in a living cell,e.g., in the cytosol, in the nucleus, in an intracellular organelle, inthe plasma membrane, or in an intracellular membrane of the cell.

Biological cells which are suitable for use in a subject screening assayinclude, but are not limited to, primary cultures of mammalian cells,transgenic (non-human) organisms and mammalian tissue. Cells inscreening assays may be dissociated either immediately or after primaryculture. Cell types include, but are not limited to white blood cells(e.g. leukocytes), hepatocytes, pancreatic beta-cells, neurons, smoothmuscle cells, intestinal epithelial cells, cardiac myocytes, glialcells, and the like.

Biological cells which are suitable for use in a subject screening assayinclude cultured cell lines (e.g., immortalized cell lines).Representative suitable cultured cell lines derived from humans andother mammals include, but are not limited to, HeLa cells (e.g.,American Type Culture Collection (ATCC) No. CCL-2), CHO cells (e.g.,ATCC Nos. CRL9618, CCL61, CRL9096), 293 cells (e.g., ATCC No. CRL-1573),Vero cells, NIH 3T3 cells (e.g., ATCC No. CRL-1658), Huh-7 cells, BHKcells (e.g., ATCC No. CCL10), PC12 cells (ATCC No. CRL1721), COS cells,COS-7 cells (ATCC No. CRL1651), RAT1 cells, mouse L cells (ATCC No.CCLI.3), human embryonic kidney (HEK) cells (ATCC No. CRL1573), HLHepG2cells, and the like.

In some embodiments, the cell is a neuronal cell or a neuronal-likecell. The cells can be of human, non-human primate, mouse, or ratorigin, or derived from a mammal other than a human, non-human primate,rat, or mouse. Suitable cell lines include, but are not limited to, ahuman glioma cell line, e.g., SVGp12 (ATCC CRL-8621), CCF-STTG1 (ATCCCRL-1718), SW 1088 (ATCC HTB-12), SW 1783 (ATCC HTB-13), LLN-18 (ATCCCRL-2610), LNZTA3WT4 (ATCC CRL-11543), LNZTA3WT11 (ATCC CRL-11544),U-138 MG (ATCC HTB-16), U-87 MG (ATCC HTB-14), H4 (ATCC HTB-148), andLN-229 (ATCC CRL-2611); a human medulloblastoma-derived cell line, e.g.,D342 Med (ATCC HTB-187), Daoy (ATCC HTB-186), D283 Med (ATCC HTB-185); ahuman tumor-derived neuronal-like cell, e.g., PFSK-1 (ATCC CRL-2060),SK-N-DZ (ATCCCRL-2149), SK-N-AS (ATCC CRL-2137), SK-N-FI (ATCCCRL-2142), IMR-32 (ATCC CCL-127), etc.; a mouse neuronal cell line,e.g., BC3H1 (ATCC CRL-1443), EOC1 (ATCC CRL-2467), C8-D30 (ATCCCRL-2534), C8-S (ATCC CRL-2535), Neuro-2a (ATCC CCL-131), NB41A3 (ATCCCCL-147), SW10 (ATCC CRL-2766), NG108-15 (ATCC HB-12317); a rat neuronalcell line, e.g., PC-12 (ATCC CRL-1721), CTX TNA2 (ATCC CRL-2006), C6(ATCC CCL-107), F98 (ATCC CRL-2397), RG2 (ATCC CRL-2433), B35 (ATCCCRL-2754), R3 (ATCC CRL-2764), SCP (ATCC CRL-1700), OA1 (ATCC CRL-6538).

In some embodiments, the readout for an effect on the activity of theligand-binding, light regulated polypeptide is a direct measure of theactivity of the ligand-binding, light regulated polypeptide. A directeffect on the ligand-binding, light regulated polypeptide is detectedusing an assay appropriate to the particular protein. For example, wherethe ligand-binding, light regulated is an enzyme, any of a variety ofassays can be used to detect enzymatic activity, and therefore to detectan effect of the test agent on enzymatic activity. As another example,where the ligand-binding, light regulated polypeptide is an ion channel,the effect, if any, of the test agent on the activity of the ion channelis in some embodiments detected by detecting a change in theintracellular concentration of an ion. A change in the intracellularconcentration of an ion can be detected using an indicator appropriateto the ion whose influx is controlled by the channel. For example, wherethe ion channel is a potassium ion channel, a potassium-detecting dye isused; where the ion channel is a calcium ion channel, acalcium-detecting dye is used; etc.

In other embodiments, the readout for an effect on the activity of theligand-binding, light regulated polypeptide is an effect on a secondpolypeptide whose activity is affected by the ligand-binding, lightregulated polypeptide. For example, where the ligand-binding, lightregulated polypeptide is an ion channel that controls influx ofpotassium into the cell, where an influx of potassium into the cellgenerates a voltage across the membrane, the effect of a test agent onactivity of the ligand-binding, light regulated polypeptide can bedetected by detecting a voltage generated across the membrane. In someembodiments, where the ligand-binding, light regulated polypeptide is anion channel that controls influx of potassium into the cell, the ionchannel controls opening of a calcium channel. In these embodiments, acalcium-sensitive dye is used to detect an effect of the test agent onthe activity of the ligand-binding, light regulated ion channel.

Suitable voltage-sensitive dyes include, but are not limited to,merocyanine-oxazolone dyes (e.g., NK2367); merocyanine-rhodanine dyes(e.g., NK2495, NK2761, NK2776, NK3224, and NK3225); oxonol dyes (e.g.,RH155, RH479, RH482, RH1691, RH1692, and RH1838); styryl dyes (e.g.,RH237, RH414, RH421, RH437, RH461, RH795, JPW1063, JPW3028, di-4-ANEPPS,di-9-ANEPPS, di-2-ANEPEQ, di-12-ANEPEQ, di-8-ANEPPQ, and di-12-ANEPPQ);and the like.

Suitable intracellular K⁺ ion-detecting dyes include, but are notlimited to, K⁺-binding benzofuran isophthalate and the like.

Suitable intracellular Ca²⁺ ion-detecting dyes include, but are notlimited to, fura-2, bis-fura 2, indo-1, Quin-2, Quin-2 AM,Benzothiaza-1, Benzothiaza-2, indo-5F, Fura-FF, BTC, Mag-Fura-2,Mag-Fura-5, Mag-Indo-1, fluo-3, rhod-2, fura-4F, fura-5F, fura-6F,fluo-4, fluo-5F, fluo-5N, Oregon Green 488 BAPTA, Calcium Green,Calcein, Fura-C18, Calcium Green-C18, Calcium Orange, Calcium Crimson,Calcium Green-5N, Magnesium Green, Oregon Green 488 BAPTA-1, OregonGreen 488 BAPTA-2, X-rhod-1, Fura Red, Rhod-5F, Rhod-5N, X-Rhod-5N,Mag-Rhod-2, Mag-X-Rhod-1, Fluo-5N, Fluo-5F, Fluo-4FF, Mag-Fluo-4,Aequorin, dextran conjugates or any other derivatives of any of thesedyes, and others (see, e.g., the catalog or Internet site for MolecularProbes, Eugene, see, also, Nuccitelli, ed., Methods in Cell Biology,Volume 40: A Practical Guide to the Study of Calcium in Living Cells,Academic Press (1994); Lambert, ed., Calcium Signaling Protocols(Methods in Molecular Biology Volume 114), Humana Press (1999); W. T.Mason, ed., Fluorescent and Luminescent Probes for Biological Activity.A Practical Guide to Technology for Quantitative Real-Time Analysis,Second Ed, Academic Press (1999); Calcium Signaling Protocols (Methodsin Molecular Biology), 2005, D. G. Lamber, ed., Humana Press.).

In particular embodiments of interest, a subject screening method isuseful for identifying agents that reduce or relieve pain, e.g., agentsthat bind an opioid receptor, where the screening method involvesdetecting an effect, if any, of the test agent on the activity of apotassium channel or calcium channel that is regulated by the opioidreceptor. In other embodiments, a subject screening method is useful foridentifying agents that are selective for a particular receptor type orsubtype, where the screening method involves determining the effect ofthe agent on a first subtype and a second subtype, where an effect onthe first subtype, and a reduced effect (or substantially no effect) onthe second subtype indicates selectivity of the test agent for the firstsubtype.

Therapeutic Applications

A subject synthetic regulator of protein function is suitable for use ina variety of therapeutic applications, which are also provided. In someembodiments, a subject synthetic regulator of protein function is usefulin restoring light sensitivity to a retina that has reduced lightsensitivity. In other embodiments, a subject synthetic regulator ofprotein function is useful as a local anesthetic. In other embodiments,a subject synthetic regulator is useful as an anti-convulsant, e.g., inthe treatment of epilepsy.

Restoring Light Sensitivity to a Retina

The present invention provides a method for restoring light sensitivityto a retina, or conferring light sensitivity to a cell in the eye, themethod generally involving administering to an individual in needthereof an effective amount of a subject synthetic regulator of proteinfunction locally, e.g., in or around the eye.

A subject synthetic regulator that is suitable for this applicationcomprises a ligand that confers light sensitivity on one or more cellsin the eye, e.g., retinal pigment epithelial cells; and cells disposedin the neurosensory retina, for example, photoreceptor cells and Muellercells. A pharmaceutical composition comprising a subject syntheticregulator is administered in or around the eye; the synthetic regulatorattaches to a protein in a cell in the eye, and confers lightsensitivity to the cell. Suitable pharmaceutical compositions aredescribed in detail below. For example, the synthetic regulator canconfer light sensitivity on a retinal ganglion.

A pharmaceutical composition comprising a subject synthetic regulatorthat confers light sensitivity on a cell can be delivered to the eyethrough a variety of routes. A subject pharmaceutical composition may bedelivered intraocularly, by topical application to the eye or byintraocular injection into, for example the vitreous or subretinal(interphotoreceptor) space. Alternatively, a subject pharmaceuticalcomposition may be delivered locally by insertion or injection into thetissue surrounding the eye. A subject pharmaceutical composition may bedelivered systemically through an oral route or by subcutaneous,intravenous or intramuscular injection. Alternatively, a subjectpharmaceutical composition may be delivered by means of a catheter or bymeans of an implant, wherein such an implant is made of a porous,non-porous or gelatinous material, including membranes such as silasticmembranes or fibers, biodegradable polymers, or proteinaceous material.A subject pharmaceutical composition can be administered prior to theonset of the condition, to prevent its occurrence, for example, duringsurgery on the eye, or immediately after the onset of the pathologicalcondition or during the occurrence of an acute or protracted condition.

The effects of therapy for an ocular disorder as described herein can beassessed in a variety of ways, using methods known in the art. Forexample, the subject's vision can be tested according to conventionalmethods. Such conventional methods include, but are not necessarilylimited to, electroretinogram (ERG), focal ERG, tests for visual fields,tests for visual acuity, ocular coherence tomography (OCT), Fundusphotography, Visual Evoked Potentials (VEP) and Pupillometry. Ingeneral, the invention provides for maintenance of a subject's vision(e.g., prevention or inhibition of vision loss of further vision lossdue to photoreceptor degeneration), slows progression of vision loss, orin some embodiments, provides for improved vision relative to thesubject's vision prior to therapy.

Exemplary conditions of particular interest which are amenable totreatment according to the methods of the invention include, but are notnecessarily limited to, diabetic retinopathy, age-related maculardegeneration (AMD or ARMD) (wet form); dry AMD; retinopathy ofprematurity; retinitis pigmentosa (RP); diabetic retinopathy; andglaucoma, including open-angle glaucoma (e.g., primary open-angleglaucoma), angle-closure glaucoma, and secondary glaucomas (e.g.,pigmentary glaucoma, pseudoexfoliative glaucoma, and glaucomas resultingfrom trauma and inflammatory diseases).

Further exemplary conditions amenable to treatment according to theinvention include, but are not necessarily limited to, retinaldetachment, age-related or other maculopathies, photic retinopathies,surgery-induced retinopathies, toxic retinopathies, retinopathy ofprematurity, retinopathies due to trauma or penetrating lesions of theeye, inherited retinal degenerations, surgery-induced retinopathies,toxic retinopathies, retinopathies due to trauma or penetrating lesionsof the eye.

Specific exemplary inherited conditions of interest for treatmentaccording to the invention include, but are not necessarily limited to,Bardet-Biedl syndrome (autosomal recessive); Congenital amaurosis(autosomal recessive); Cone or cone-rod dystrophy (autosomal dominantand X-linked forms); Congenital stationary night blindness (autosomaldominant, autosomal recessive and X-linked forms); Macular degeneration(autosomal dominant and autosomal recessive forms); Optic atrophy,autosomal dominant and X-linked forms); Retinitis pigmentosa (autosomaldominant, autosomal recessive and X-linked forms); Syndromic or systemicretinopathy (autosomal dominant, autosomal recessive and X-linkedforms); and Usher syndrome (autosomal recessive).

Local Anesthetic

The present invention provides a method of reducing or preventing painin an individual, the method generally involving: a) administering to anindividual in need thereof an effective amount of a subject syntheticregulator of protein function, where the synthetic regulator of proteinfunction comprises a ligand that blocks a pain response or a painsignal, where the synthetic regulator binds to receptor or a channel,forming complex between the synthetic regulator and the receptor orchannel; and b) exposing the receptor/regulator complex orchannel/regulatory complex to a wavelength of light that provides forbinding of the ligand to the receptor or channel. For example, in someembodiments, the protein is a cation channel, and the syntheticregulator binds to the cation channel, forming a cationchannel/regulator complex, where the channel/regulator complex isexposed to a wavelength of light that provides for blocking of thechannel, e.g., a Na⁺ channel, an N-type Ca²⁺ channel, etc.

An “effective amount” of a subject synthetic regulator is an amount thatis effective to reduce pain by at least 30%, 40%, 60%, 70%, 80%, 90% or100% for a period of time of from about 15 minutes to 5 days, e.g., fromabout 15 minutes to about 30 minutes, from about 30 minutes to about 60minutes, from about 1 hour to about 4 hours, from about 4 hours to about8 hours, from about 8 hours to about 16 hours, from about 16 hours toabout 24 hours, from about 24 hours to about 36 hours, from about 36hours to about 48 hours, from about 48 hours to about 3 days, or fromabout 3 days to about 5 days. The effectiveness of a subject syntheticregulator in treating nociceptive pain can be determined by observingone or more clinical symptoms or physiological indicators associatedwith nociceptive pain.

In these embodiments, a suitable synthetic regulator includes one thatcomprises, as a ligand, an opioid analgesic. Suitable ligands include,but are not limited to, morphine, oxycodone, fentanyl, pentazocine,hydromorphone, meperidine, methadone, levorphanol, oxymorphone,levallorphan, codeine, dihydrocodeine, hydrocodone, propoxyphene,nalmefene, nalorphine, naloxone, naltrexone, buprenorphine, butorphanol,nalbuphine, and pentazocine. In other embodiments, a suitable syntheticregulator comprises a ligand moiety selected from lidocaine, novocaine,xylocalne, lignocaine, novocaine, carbocaine, etidocaine, tetracaine,procaine, prontocaine, prilocalne, bupivacaine, cinchocaine,mepivacaine, quinidine, flecamide, procaine,N—[[2′-(aminosulfonyl)biphenyl-4-yl]methyl]-N′-(2,2′-bithien-5-ylmethyl)succinamide(BPBTS), QX-314, saxitoxin, tetrodotoxin, and a type III conotoxin.

The present invention provides pharmaceutical compositions comprising asubject synthetic regulator. In some embodiments, the pharmaceuticalcomposition is suitable for administering to an individual in need of alocal anesthetic. Individuals in need of a local anesthetic include anindividual who is about to undergo a surgical procedure, and anindividual who has undergone a surgical procedure within the last 5minutes to within the last 72 hours. Individuals in need of a localanesthetic further include individuals having a wound, e.g., asuperficial wound.

A pharmaceutical composition comprising a subject synthetic regulatormay be administered to a patient alone, or in combination with othersupplementary active agents. The pharmaceutical compositions may bemanufactured using any of a variety of processes, including, withoutlimitation, conventional mixing, dissolving, granulating, dragee-making,levigating, emulsifying, encapsulating, entrapping, and lyophilizing.The pharmaceutical composition can take any of a variety of formsincluding, without limitation, a sterile solution, suspension, emulsion,lyophilisate, tablet, pill, pellet, capsule, powder, syrup, elixir orany other dosage form suitable for administration.

A pharmaceutical composition comprising a subject synthetic regulatorcan optionally include a pharmaceutically acceptable carrier(s) thatfacilitate processing of an active ingredient into pharmaceuticallyacceptable compositions. As used herein, the term “pharmacologicallyacceptable carrier” refers to any carrier that has substantially no longterm or permanent detrimental effect when administered and encompassesterms such as “pharmacologically acceptable vehicle, stabilizer,diluent, auxiliary or excipient.” Such a carrier generally is mixed withan active compound, or permitted to dilute or enclose the activecompound and can be a solid, semi-solid, or liquid agent. It isunderstood that the active ingredients can be soluble or can bedelivered as a suspension in the desired carrier or diluent. Any of avariety of pharmaceutically acceptable carriers can be used including,without limitation, aqueous media such as, e.g., distilled, deionizedwater, saline; solvents; dispersion media; coatings; antibacterial andantifungal agents; isotonic and absorption delaying agents; or any otherinactive ingredient. Selection of a pharmacologically acceptable carriercan depend on the mode of administration. Except insofar as anypharmacologically acceptable carrier is incompatible with the activeingredient, its use in pharmaceutically acceptable compositions iscontemplated. Non-limiting examples of specific uses of suchpharmaceutical carriers can be found in “Pharmaceutical Dosage Forms andDrug Delivery Systems” (Howard C. Ansel et al., eds., LippincottWilliams & Wilkins Publishers, 7^(th) ed. 1999); “Remington: The Scienceand Practice of Pharmacy” (Alfonso R. Gennaro ed., Lippincott, Williams& Wilkins, 20^(th) 2000); “Goodman & Gilman's The Pharmacological Basisof Therapeutics” Joel G. Hardman et al., eds., McGraw-Hill Professional,10.sup.th ed. 2001); and “Handbook of Pharmaceutical Excipients”(Raymond C. Rowe et al., APhA Publications, 4^(th) edition 2003).

A subject pharmaceutical composition can optionally include, withoutlimitation, other pharmaceutically acceptable components, including,without limitation, buffers, preservatives, tonicity adjusters, salts,antioxidants, physiological substances, pharmacological substances,bulking agents, emulsifying agents, wetting agents, sweetening orflavoring agents, and the like. Various buffers and means for adjustingpH can be used to prepare a pharmaceutical composition disclosed in thepresent specification, provided that the resulting preparation ispharmaceutically acceptable. Such buffers include, without limitation,acetate buffers, citrate buffers, phosphate buffers, neutral bufferedsaline, phosphate buffered saline and borate buffers. It is understoodthat acids or bases can be used to adjust the pH of a composition asneeded. Pharmaceutically acceptable antioxidants include, withoutlimitation, sodium metabisulfite, sodium thiosulfate, acetylcysteine,butylated hydroxyanisole and butylated hydroxytoluene. Usefulpreservatives include, without limitation, benzalkonium chloride,chlorobutanol, thimerosal, phenylmercuric acetate, phenylmercuricnitrate and a stabilized oxy chloro composition, for example, PURITE™.Tonicity adjustors suitable for inclusion in a subject pharmaceuticalcomposition include, without limitation, salts such as, e.g., sodiumchloride, potassium chloride, mannitol or glycerin and otherpharmaceutically acceptable tonicity adjustor. It is understood thatthese and other substances known in the art of pharmacology can beincluded in a subject pharmaceutical composition.

Some examples of materials which can serve aspharmaceutically-acceptable carriers include: (1) sugars, such aslactose, glucose and sucrose; (2) starches, such as corn starch andpotato starch; (3) cellulose, and its derivatives, such as sodiumcarboxymethyl cellulose, ethyl cellulose and cellulose acetate; (4)powdered tragacanth; (5) malt; (6) gelatin; (7) talc; (8) excipients,such as cocoa butter and suppository waxes; (9) oils, such as peanutoil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil andsoybean oil; (10) glycols, such as propylene glycol; (11) polyols, suchas glycerin, sorbitol, mannitol, and polyethylene glycol; (12) esters,such as ethyl oleate and ethyl laurate; (13) agar; (14) bufferingagents, such as magnesium hydroxide and aluminum hydroxide; (15) alginicacid; (16) pyrogen-free water; (17) isotonic saline; (18) Ringer'ssolution; (19) ethyl alcohol; (20) pH buffered solutions; (21)polyesters, polycarbonates and/or polyanhydrides; and (22) othernon-toxic compatible substances employed in pharmaceutical formulations.

Routes of administration suitable for the methods of the inventioninclude both systemic and local administration. In some embodiments, asubject pharmaceutical composition comprising a subject syntheticregulator is administered locally. As non-limiting examples, apharmaceutical composition useful for treating nociceptive pain can beadministered orally; by subcutaneous pump; by dermal patch; byintravenous, subcutaneous or intramuscular injection; by topical drops,creams, gels, sprays, or ointments; as an implanted or injected extendedrelease formulation; as a bioerodable or non-bioerodable deliverysystem; by subcutaneous minipump or other implanted device; byintrathecal pump or injection; or by epidural injection. In someembodiments, a subject pharmaceutical composition comprising a subjectsynthetic regulator is administered sublingually. In some embodiments, asubject pharmaceutical composition comprising a subject syntheticregulator is administered topically to gum tissue. In some embodiments,a subject pharmaceutical composition comprising a subject syntheticregulator is injected into gum tissue. In some embodiments, a subjectpharmaceutical composition comprising a subject synthetic regulator isadministered topically to the skin. In some embodiments, a subjectpharmaceutical composition comprising a subject synthetic regulator isadministered at or near a site of a surgical incision. In someembodiments, a subject pharmaceutical composition comprising a subjectsynthetic regulator is administered intramuscularly at the site of asurgical incision. For example, in some embodiments, a subjectpharmaceutical composition comprising a subject synthetic regulator isadministered at a surgical site, and before the surgical wound isclosed, the synthetic regulator/target protein complex is exposed tolight of a wavelength that induces binding of the ligand to the protein.In some embodiments, a subject pharmaceutical composition isadministered (e.g., injected) at or near a nerve. Thus, in someembodiments, a subject pharmaceutical composition is formulated forinjection at or near a nerve. For example, for oral surgery, a subjectpharmaceutical composition is injected at or near a nerve in gum tissue.

In some embodiments, a subject pharmaceutical composition comprising asubject synthetic regulator is administered just before surgery, e.g.,from about 1 minute to about 2 hours before surgery, e.g., from about 1minute to about 5 minutes, from about 5 minutes to about 15 minutes fromabout 15 minutes to about 30 minutes, from about 30

A subject synthetic regulator comprising a ligand that provides for painprevention is suitable for preventing or reducing pain in an individualin need thereof. Individuals in need of treatment with a subjectsynthetic regulator comprising a ligand that provides for painprevention include individuals who are about to undergo surgery, e.g.,individuals who are scheduled to undergo a surgical procedure in thenext 5 minutes to 72 hours; individuals who are undergoing a surgicalprocedure; and individuals who have undergone a surgical procedurewithin the previous 5 minutes to 1 hour. Thus, individuals sufferingfrom post-operative pain are suitable for treatment. A subject syntheticregulator comprising a ligand that provides for pain prevention is alsosuitable for preventing or reducing pain in an individual having awound, e.g., a superficial wound.

Anti-Convulsant Applications

In some embodiments, a subject synthetic regulator comprises, as aligand, a ligand for a sodium channel, a potassium channel, or a GABAreceptor, where the ligand functions as an anti-convulsant. In someembodiments, the synthetic regulator is administered in a pharmaceuticalcomposition, as described supra and infra.

Pharmaceutical Compositions

A subject synthetic regulator can be formulated with one or morepharmaceutically acceptable excipients. A wide variety ofpharmaceutically acceptable excipients are known in the art and need notbe discussed in detail herein. Pharmaceutically acceptable excipientshave been amply described in a variety of publications, including, forexample, A. Gennaro (2000) “Remington: The Science and Practice ofPharmacy,” 20th edition, Lippincott, Williams, & Wilkins; Pharmaceutical57-th ed., Dosage Forms and Drug Delivery Systems (1999) H. C. Ansel etal., eds. Lippincott, Williams, & Wilkins; and Handbook ofPharmaceutical Excipients (2000) A. H. Kibbe et al., eds., 3^(rd) ed.Amer. Pharmaceutical Assoc.

The pharmaceutically acceptable excipients, such as vehicles, adjuvants,carriers or diluents, are readily available to the public. Moreover,pharmaceutically acceptable auxiliary substances, such as pH adjustingand buffering agents, tonicity adjusting agents, stabilizers, wettingagents and the like, are readily available to the public

In the subject methods, a subject synthetic regulator may beadministered to the host using any convenient means capable of resultingin the desired reduction in disease condition or symptom. Thus, asubject synthetic regulator can be incorporated into a variety offormulations for therapeutic administration. More particularly, asubject synthetic regulator can be formulated into pharmaceuticalcompositions by combination with appropriate pharmaceutically acceptablecarriers or diluents, and may be formulated into preparations in solid,semi-solid, liquid or gaseous forms, such as tablets, capsules, powders,granules, ointments, solutions, suppositories, injections, inhalants andaerosols.

A subject synthetic regulator can be used alone or in combination withappropriate additives to make tablets, powders, granules or capsules,for example, with conventional additives, such as lactose, mannitol,corn starch or potato starch; with binders, such as crystallinecellulose, cellulose derivatives, acacia, corn starch or gelatins; withdisintegrators, such as corn starch, potato starch or sodiumcarboxymethylcellulose; with lubricants, such as talc or magnesiumstearate; and if desired, with diluents, buffering agents, moisteningagents, preservatives and flavoring agents. Such preparations can beused for oral administration.

A subject synthetic regulator can be formulated into preparations forinjection by dissolving, suspending or emulsifying them in an aqueous ornonaqueous solvent, such as vegetable or other similar oils, syntheticaliphatic acid glycerides, esters of higher aliphatic acids or propyleneglycol; and if desired, with conventional additives such assolubilizers, isotonic agents, suspending agents, emulsifying agents,stabilizers and preservatives. Formulations suitable for injection canbe administered by an intravitreal, intraocular, intramuscular,subcutaneous, sublingual, or other route of administration, e.g.,injection into the gum tissue or other oral tissue. Such formulationsare also suitable for topical administration.

A subject synthetic regulator can be utilized in aerosol formulation tobe administered via inhalation. A subject synthetic regulator can beformulated into pressurized acceptable propellants such asdichlorodifluoromethane, propane, nitrogen and the like.

Furthermore, a subject synthetic regulator can be made intosuppositories by mixing with a variety of bases such as emulsifyingbases or water-soluble bases. A subject synthetic regulator can beadministered rectally via a suppository. The suppository can includevehicles such as cocoa butter, carbowaxes and polyethylene glycols,which melt at body temperature, yet are solidified at room temperature.

Unit dosage forms for oral or rectal administration such as syrups,elixirs, and suspensions may be provided wherein each dosage unit, forexample, teaspoonful, tablespoonful, tablet or suppository, contains apredetermined amount of the composition containing one or moreinhibitors. Similarly, unit dosage forms for injection or intravenousadministration may comprise a subject synthetic regulator in acomposition as a solution in sterile water, normal saline or anotherpharmaceutically acceptable carrier.

The term “unit dosage form,” as used herein, refers to physicallydiscrete units suitable as unitary dosages for human and animalsubjects, each unit containing a predetermined quantity of a subjectsynthetic regulator calculated in an amount sufficient to produce thedesired effect in association with a pharmaceutically acceptablediluent, carrier or vehicle. The specifications for a subject syntheticregulator depend on the particular compound employed and the effect tobe achieved, and the pharmacodynamics associated with each compound inthe host.

A subject synthetic regulator can be administered as injectables.Injectable compositions are prepared as liquid solutions or suspensions;solid forms suitable for solution in, or suspension in, liquid vehiclesprior to injection may also be prepared. The preparation may also beemulsified or the active ingredient encapsulated in liposome vehicles.

In some embodiments, a subject synthetic regulator is delivered by acontinuous delivery system. The term “continuous delivery system” isused interchangeably herein with “controlled delivery system” andencompasses continuous (e.g., controlled) delivery devices (e.g., pumps)in combination with catheters, injection devices, and the like, a widevariety of which are known in the art.

Mechanical or electromechanical infusion pumps can also be suitable foruse with the present invention. Examples of such devices include thosedescribed in, for example, U.S. Pat. Nos. 4,692,147; 4,360,019;4,487,603; 4,360,019; 4,725,852; 5,820,589; 5,643,207; 6,198,966; andthe like. In general, delivery of a subject synthetic regulator can beaccomplished using any of a variety of refillable, pump systems. Pumpsprovide consistent, controlled release over time. In some embodiments,the agent is in a liquid formulation in a drug-impermeable reservoir,and is delivered in a continuous fashion to the individual.

In one embodiment, the drug delivery system is an at least partiallyimplantable device. The implantable device can be implanted at anysuitable implantation site using methods and devices well known in theart. An implantation site is a site within the body of a subject atwhich a drug delivery device is introduced and positioned. Implantationsites include, but are not necessarily limited to a subdermal,subcutaneous, intramuscular, or other suitable site within a subject'sbody. Subcutaneous implantation sites are used in some embodimentsbecause of convenience in implantation and removal of the drug deliverydevice.

In some embodiments, the drug delivery device is an implantable device.The drug delivery device can be implanted at any suitable implantationsite using methods and devices well known in the art. As noted infra, animplantation site is a site within the body of a subject at which a drugdelivery device is introduced and positioned. Implantation sitesinclude, but are not necessarily limited to a subdermal, subcutaneous,intramuscular, or other suitable site within a subject's body.

In some embodiments, a subject synthetic regulator is delivered using animplantable drug delivery system, e.g., a system that is programmable toprovide for administration of the agent. Exemplary programmable,implantable systems include implantable infusion pumps. Exemplaryimplantable infusion pumps, or devices useful in connection with suchpumps, are described in, for example, U.S. Pat. Nos. 4,350,155;5,443,450; 5,814,019; 5,976,109; 6,017,328; 6,171,276; 6,241,704;6,464,687; 6,475,180; and 6,512,954. A further exemplary device that canbe adapted for the present invention is the Synchromed infusion pump(Medtronic).

Suitable excipient vehicles are, for example, water, saline, dextrose,glycerol, ethanol, or the like, and combinations thereof. In addition,if desired, the vehicle may contain minor amounts of auxiliarysubstances such as wetting or emulsifying agents or pH buffering agents.Actual methods of preparing such dosage forms are known, or will beapparent, to those skilled in the art. See, e.g., Remington'sPharmaceutical Sciences, Mack Publishing Company, Easton, Pa., 17thedition, 1985. The composition or formulation to be administered will,in any event, contain a quantity of the synthetic regulator adequate toachieve the desired state in the subject being treated.

The pharmaceutically acceptable excipients, such as vehicles, adjuvants,carriers or diluents, are readily available to the public. Moreover,pharmaceutically acceptable auxiliary substances, such as pH adjustingand buffering agents, tonicity adjusting agents, stabilizers, wettingagents and the like, are readily available to the public.

Ophthalmic Formulations

In some embodiments, a subject pharmaceutical composition comprises asubject synthetic regulator formulated for ophthalmic application. Forophthalmic application, solutions or medicaments are often preparedusing a physiological saline solution as a major vehicle. Ophthalmicsolutions should be maintained at a comfortable pH with an appropriatebuffer system. The formulations may also contain conventional,pharmaceutically acceptable preservatives, stabilizers and surfactants.

Preservatives that may be used in a subject pharmaceutical compositioninclude, but are not limited to, benzalkonium chloride, chlorobutanol,thimerosal, phenylmercuric acetate and phenylmercuric nitrate. A usefulsurfactant is, for example, Tween 80. Likewise, various useful vehiclesmay be used in the ophthalmic preparations of the present invention.These vehicles include, but are not limited to, polyvinyl alcohol,povidone, hydroxypropyl methyl cellulose, poloxamers, carboxymethylcellulose, hydroxyethyl cellulose and purified water.

Tonicity adjustors may be added as needed or convenient. They include,but are not limited to, salts, particularly sodium chloride, potassiumchloride, mannitol and glycerin, or any other suitable ophthalmicallyacceptable tonicity adjustor.

Various buffers and means for adjusting pH may be used so long as theresulting preparation is ophthalmically acceptable. Accordingly, buffersinclude acetate buffers, citrate buffers, phosphate buffers and boratebuffers. Acids or bases may be used to adjust the pH of theseformulations as needed.

An ophthalmically acceptable antioxidant for use in the presentinvention includes, but is not limited to, sodium metabisulfite, sodiumthiosulfate, acetylcysteine, butylated hydroxyanisole and butylatedhydroxytoluene.

Other excipient components which can be included in the ophthalmicpreparations are chelating agents. A useful chelating agent is edetatedisodium (ethylenediamine tetraacetate disodium; EDTA-disodium),although other chelating agents may also be used in place or inconjunction with it.

Dosages

In general, a subject synthetic regulator is administered in an amountof from about 10 ng to about 10 mg per dose, e.g., from about 10 ng toabout 20 ng, from about 20 ng to about 25 ng, from about 25 ng to about50 ng, from about 50 ng to about 75 mg, from about 75 ng to about 100ng, from about 100 ng to about 125 ng, from about 125 ng to about 150ng, from about 150 ng to about 175 ng, from about 175 ng to about 200ng, from about 200 ng to about 225 ng, from about 225 ng to about 250ng, from about 250 ng to about 300 ng, from about 300 ng to about 350ng, from about 350 ng to about 400 ng, from about 400 ng to about 450ng, from about 450 ng to about 500 ng, from about 500 ng to about 750ng, from about 750 ng to about 1 mg, from about 1 mg to about 5 mg, orfrom about 5 mg to about 10 mg per dose. In some embodiments, the amountof a subject synthetic regulator per dose is determined on a per bodyweight basis.

Those of skill will readily appreciate that dose levels can vary as afunction of the specific compound, the severity of the symptoms and thesusceptibility of the subject to side effects. Preferred dosages for agiven compound are readily determinable by those of skill in the art bya variety of means.

In some embodiments, multiple doses of a subject synthetic regulator areadministered. The frequency of administration of a subject syntheticregulator can vary depending on any of a variety of factors, e.g.,severity of the symptoms, etc. For example, in some embodiments, asubject synthetic regulator is administered once per month, twice permonth, three times per month, every other week (qow), once per week(qw), twice per week (biw), three times per week (tiw), four times perweek, five times per week, six times per week, every other day (qod),daily (qd), twice a day (qid), or three times a day (tid). As discussedabove, in some embodiments, a subject synthetic regulator isadministered continuously.

The duration of administration of a subject synthetic regulator, e.g.,the period of time over which a subject synthetic regulator isadminister, can vary, depending on any of a variety of factors, e.g.,patient response, etc. For example, a subject synthetic regulator can beadministered over a period of time ranging from about one day to aboutone week, from about two weeks to about four weeks, from about one monthto about two months, from about two months to about four months, fromabout four months to about six months, from about six months to abouteight months, from about eight months to about 1 year, from about 1 yearto about 2 years, or from about 2 years to about 4 years, or more.

Routes of Administration

A subject synthetic regulator is administered to an individual using anyavailable method and route suitable for drug delivery, including in vivoand ex vivo methods, as well as systemic and localized routes ofadministration.

Conventional and pharmaceutically acceptable routes of administrationinclude intranasal, intramuscular, intratracheal, subcutaneous,intradermal, topical application, intravenous, rectal, nasal, oral, andother enteral and parenteral routes of administration. Routes ofadministration may be combined, if desired, or adjusted depending uponthe agent and/or the desired effect. The subject synthetic regulator canbe administered in a single dose or in multiple doses.

A subject synthetic regulator can be administered to a host using anyavailable conventional methods and routes suitable for delivery ofconventional drugs, including systemic or localized routes. In general,routes of administration contemplated by the invention include, but arenot necessarily limited to, enteral, parenteral, topical, intraorbital,and intravitreous routes.

Parenteral routes of administration other than inhalation administrationinclude, but are not necessarily limited to, topical, transdermal,subcutaneous, intramuscular, intraorbital, intracapsular, intraspinal,intrasternal, and intravenous routes, i.e., any route of administrationother than through the alimentary canal. Parenteral administration canbe carried to effect systemic or local delivery of the subject syntheticregulator. Where systemic delivery is desired, administration typicallyinvolves invasive or systemically absorbed topical or mucosaladministration of pharmaceutical preparations.

A subject synthetic regulator can also be delivered to the subject byenteral administration. Enteral routes of administration include, butare not necessarily limited to, oral and rectal (e.g., using asuppository) delivery.

Methods of administration of the subject synthetic regulator through theskin or mucosa include, but are not necessarily limited to, topicalapplication of a suitable pharmaceutical preparation, transdermaltransmission, injection and epidermal administration. For transdermaltransmission, absorption promoters or iontophoresis are suitablemethods. Iontophoretic transmission may be accomplished usingcommercially available “patches” which deliver their productcontinuously via electric pulses through unbroken skin for periods ofseveral days or more.

Kits

Also provided are kits that find use in practicing the subject methods,as described above. For example, in some embodiments, kits forpracticing the subject methods include at least a synthetic regulator,as described above. In other embodiments, kits for practicing thesubject methods include at least a light-regulated polypeptide asdescribed above. In some embodiments, a subject kit includes apolypeptide and a subject synthetic regulator. In other embodiments, asubject kit includes a polypeptide with a subject synthetic regulator instable association with the polypeptide. In other embodiments, kits forpracticing the subject methods include at least a test cell as describedabove, or elements for constructing the same, e.g., expression vectors,etc. In some embodiments, a subject kit includes a cell (e.g., aeukaryotic cell or a prokaryotic cell), where the cell produces aprotein to be controlled, directly or indirectly, by a subject syntheticregulator; and a subject synthetic regulator. Furthermore, additionalreagents that are required or desired in the protocol to be practicedwith the kit components may be present, which additional reagentsinclude, but are not limited to: aqueous media, culture media, and thelike. The kits may also include reference or control elements, e.g.,that provide calibration signals or values for use in assessing theobserved signal generated by an assay performed with the kit components.The kit components may be present in separate containers, or one or moreof the components may be present in the same container, where thecontainers may be storage containers and/or containers that are employedduring the assay for which the kit is designed.

In addition to the above components, the subject kits may furtherinclude instructions for practicing the subject methods. Theseinstructions may be present in the subject kits in a variety of forms,one or more of which may be present in the kit. One form in which theseinstructions may be present is as printed information on a suitablemedium or substrate, e.g., a piece or pieces of paper on which theinformation is printed, in the packaging of the kit, in a packageinsert, etc. Yet another means would be a computer readable medium,e.g., diskette, digital versatile disc, compact disk, etc., on which theinformation has been recorded. Yet another means that may be present isa website address which may be used via the internet to access theinformation at a removed site. Any convenient means may be present inthe kits.

Systems

Also provided are systems that find use in practicing the subjectmethods, as described above. For example, in some embodiments, systemsfor practicing the subject methods include at least synthetic regulatoras described above. In other embodiments, systems for practicing thesubject methods include at least a light-regulated polypeptide asdescribed above. In other embodiments, systems for practicing thesubject methods include at least a test cell as described above.Furthermore, additional reagents that are required or desired in theprotocol to be practiced with the system components may be present,which additional reagents include, but are not limited to: aqueousmediums, culture mediums, and the like. The systems may also includereference or control elements, e.g., that provide calibration signals orvalues for use in assessing the observed signal generated by an assayperformed with the system components. The systems generally also includeone or more candidate agents.

Devices

Also provided are high throughput (HT) devices that find use inpracticing the subject methods, particularly HT embodiments thereof. Thehigh throughput devices may have any convenient configuration, andgenerally include a plurality of two or more fluid containment elementsin which assays can take place, agent administration elements and signaldetection elements. For example, representative HT devices of thesubject invention include a plate or substrate having a plurality offluid-containing wells, reagent-adding equipment responsive to acomputer for adding reagent, e.g., candidate agent, to the wells,measurement equipment for measuring at least one attribute of the sampleor cells contained by the wells (e.g., for phenotype evaluation), alight source for providing light of different wavelengths to thecontents of the wells, and moving equipment which is responsive to thecomputer for aligning one of the wells first with the reagent-addingcomponent, then with the measurement device. See, e.g., U.S. Pat. No.6,127,133. Also of interest are the devices described in U.S. Pat. Nos.6,468,736 and 5,989,835. A feature of the HT devices of the presentinvention is that they include in at least one fluid containment elementcontaining a test polypeptide, a test cell, or a test membrane asdescribed above.

EXAMPLES

The following examples are put forth so as to provide those of ordinaryskill in the art with a complete disclosure and description of how tomake and use the present invention, and are not intended to limit thescope of what the inventors regard as their invention nor are theyintended to represent that the experiments below are all or the onlyexperiments performed. Efforts have been made to ensure accuracy withrespect to numbers used (e.g. amounts, temperature, etc.) but someexperimental errors and deviations should be accounted for. Unlessindicated otherwise, parts are parts by weight, molecular weight isweight average molecular weight, temperature is in degrees Celsius, andpressure is at or near atmospheric. Standard abbreviations may be used,e.g., bp, base pair(s); kb, kilobase(s); pl, picoliter(s); s or sec,second(s); min, minute(s); h or hr, hour(s); aa, amino acid(s); kb,kilobase(s); bp, base pair(s); nt, nucleotide(s); i.m.,intramuscular(ly); i.p., intraperitoneal(ly); s.c., subcutaneous(ly);and the like.

Example 1 Light-Activated Ion Channel

Methods

Synthesis of MAL-AZO-QA

MAL-AZO-QA was synthesized in a two-step coupling procedure from thecommercially available 4,4′-azodianiline and the respective acidchlorides of the maleimide and quaternary ammonium components.

Patch Recordings from Oocytes

Xenopus oocytes were injected with 12.5-100 μg of mRNA encoding ShakerH4, with the following mutations: Δ6-46, E422C, T449V. We found that theeffects of light on channel activity were largest for the TEA bindingsite mutant T449V as compared to the wild-type channel (T449) and twoother mutants (T449Y, T449F). Devitillenized oocytes were recorded from2-10 days post-injection using standard patch-clamp methods. Foroutside-out patches, glass patch pipettes (2-4 MΩ) were filled with asolution containing (in mM) 100 KCl, 10 HEPES, 0.1 CaCl₂, 0.5 MgCl₂, and10 EGTA, while the bath contained 10 KCl, 90 NaCl, 10 HEPES, 0.1 CaCl₂,0.5 MgCl₂, and 10 EGTA. For inside-out patches, patch pipettes and bathboth contained 100 KCl, 10 HEPES, 0.1 CaCl₂, 0.5 MgCl₂, and 10 EGTA. ThepH of all solutions was 7.1. Solid MAL-AZO-QA was dissolved as aconcentrated stock solution in DMSO and stored at −20° C. until the dayof use. Stocks were diluted into oocyte bath solution to finalconcentrations of 10 μM or 100 μM. The concentration of DMSO in the bathdid not exceed 0.1%. Pulse protocols and measurements were performedwith pCLAMP software, a DigiData 1200 series interface, and a PC-505amplifier (Warner Instruments). Samples were taken at 10 kHz and thedata was filtered at 1 kHz. Patches were held at −90 mV, pulsed to −100mV for 60 msec to monitor leak, and pulsed to −20 mV for 30-100 msec at1 Hz to elicit Shaker currents. The peak of the Shaker currents wasmeasured to minimize the contribution of slow inactivation. Patches witha leak conductance >100 pS were not included in analysis.

Illumination of patches was achieved with a TILL Photonics Polychrome IImonochrometer (Applied Scientific Instrumentation, Inc.) containing a 75watt xenon short arc lamp with an output of 250-690 nm, a quartz fiberoptic cable, and an epifluorescence condenser with an achromatic lens.Discrete wavelengths of light (+/−10 nm) were focused on patches througha quartz coverslip with a Fluor 20X, 0.5 NA objective lens (Nikon).Output intensity was measured for wavelengths between 300 and 600 nm.The measured output intensities for wavelengths between 340-600 nmranged from 0.324e-8 to 6.23e-8 W/cm². Differences in light intensitiesat different wavelengths were taken into account when determining actionspectra for channel block and unblock.

Recordings from Hippocamal Neurons

Primary dissociated hippocampal cultures were prepared from E18-19Sprague-Dewey rat embryos and grown on glass cover slips inserum-containing media and incubated in 7% CO₂ in air at 37° C. Cellswere co-transfected with GFP along with the modified Shaker channeldescribed above with additional L366A and V454L mutations. Transfectionswith Lipofectamine 2000™ were performed at 12-14 DIV. About 10% of thecultured neurons appeared GFP-positive at 2-3 days after transfection.Coverslips containing the neurons were treated at 37° C. for 15 min with300 μM MAL-AZO-QA in an extracellular recording solution containing (inmM) 135 NaCl, 5 KCl, 1.2 MgCl₂, 5 HEPES, 2.5 CaCl₂, and 10 glucose at pH7.4. Patch pipettes (7-10 MΩ) were filled with 10 NaCl, 135 K-gluconate,10 HEPES, 2 MgCl₂, 2 Mg-ATP, and 1 EGTA at pH 7.4. After washout ofMAL-AZO-QA with extracellular solution, membrane potential was recordedat room temperature under whole-cell current clamp with an AXOPATCH 200Aamplifier (Axon Instruments) and filtered at 10 kHz. Initial recordingswere made at resting potential to evaluate the effects of light onspontaneous activity. In experiments where we wanted to quantify theeffect of light on firing (e.g. FIG. 4 b), cultures were treated with6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) (1 μM) and bicuculline (10μM) to silence synaptic activity. Baseline currents were adjusted to setthe membrane potential at −50 mV before depolarizing current stepsranging from 0.01-0.03 nA were applied to evoke action potentials. Cellswere irradiated with a Lambda-LS illuminator containing a 125 watt xenonarc lamp (Sutter Instruments Company), equipped with narrow bandpass(+/−10 nm) filters through a Fluor 20X, 0.5 NA objective lens (Nikon).Variability among data is expressed as mean+/−SEM, unless notedotherwise.

Results

As a starting point for engineering a light-activated channel, theShaker K⁺ channel was used because of the availability of structural andmolecular information. Sigworth, F. Quart. Rev. Biophys. 27, 1-40(1993); Laine, et al. FEBS Lett. 564, 257-263 (2004). Voltage-gated K⁺channels, including Shaker, are blocked by the binding of quaternaryammonium (QA) ions, such as tetraethylammonium (TEA), to a site in thepore-lining domain. MacKinnon & Yellen, Science 250, 276-279 (1990);Heginbotham, MacKinnon, Neuron 8, 483-491 (1992). Amino acid E422 isestimated to be 15-18 Å from the TEA binding site. Blaustein, et al.Nature Struct. Biol. 7, 309-311 (2000); Doyle, et al. Science 280, 69-77(1998); Jiang, et al. Nature 423, 33-41 (2003). Tethering of a series ofQA-containing compounds to position 422 shows that the degree of blockis critically dependent on tether length, with a 5 Å difference inlength making the difference between effective and ineffective block.Blaustein et al. (2000) supra. This information was used in designingand synthesizing a photoswitchable blocker that can be tethered onto theoutside of modified Shaker channels. The molecule, MAL-AZO-QA, consistsof a maleimide (MAL), for cysteine tethering; a QA group, to block thechannel; and an AZO group in between (FIG. 1 a). Previous studies showthat the rigid AZO moiety shortens by ˜7 Å when photoisomerized from thetrans to the cis configuration. Knoll, H. Photoisomerism of Azobenzenes.in CRC Handbook of Organic Photochemistry and Photobiology, 2^(nd) ed.(eds. Horspool, W. & Lenci, F.) 89.1-89.16 (CRC Press, Boca Raton, Fla.,2004). It was reasoned that coupling MAL-AZO-QA to a cysteine introducedat residue 422 (mutant E422C) would block channels when the compound isin the long trans form, whereas photoconversion to the cis configurationwould make the tether too short to permit block (FIG. 1 b). Hence, thetethering of MAL-AZO-QA to Shaker should introduce a new extracellulargate that can be opened and closed with appropriate wavelengths oflight.

FIGS. 1A and 1B Photoisomerization of MAL-AZO-QA gates ionic currentsthrough modified Shaker channels. (1 a) The rigid core of MAL-AZO-QA(between the a carbons flanking the AZO moiety) changes by ˜7 Å uponphotoisomerization. (1 b) MAL-AZO-QA blocks ion flow in the transconfiguration but is too short to block effectively afterphotoisomerization to the cis configuration. Diagram shows a model ofthe inner helices of the Shaker K⁺ channel, derived from the crystalstructure of the bacterial K⁺ channel MthK¹², with the dimensions ofMAL-AZO-QA drawn to scale.

The effects of MAL-AZO-QA were tested on Shaker channels expressed inXenopus oocytes. To observe the time course of channel modification,MAL-AZO-QA was applied onto the extracellular surface of the channels inoutside-out membrane patches. MAZ-AZO-QA application reduced thevoltage-gated Shaker current by >60% over 4 min (FIG. 2 a), but thelimited survival time of excised patches made it difficult to assess thefull magnitude of block. Channel block developed slowly and persistedafter washout (FIG. 2 b), consistent with covalent attachment to thechannels. Subsequent exposure to ultraviolet light partly relievedchannel block and exposure to visible light restored block. In contrast,light had no effect on channels in patches that had not been treatedwith MAL-AZO-QA.

To achieve more complete block of the channels, intact oocytes weretreated with a higher concentration of MAL-AZO-QA for 30 min and thenrecorded from inside-out patches (FIG. 2 c). In this situation,ultraviolet light unblocked as much as 1 nA of current, visible lightre-blocked the channels almost completely, and both effects nearlyreached steady-state within 5 sec under standard epifluorescenceillumination. With steady ultraviolet illumination, the channelsremained unblocked. However, in the dark unblocked channels slowly (>5min) returned to the blocked state (FIG. 2D), consistent with thermalrelaxation of the AZO moiety to the more stable trans configuration inthe absence of light. Knoll, H. Photoisomerism of Azobenzenes. in CRCHandbook of Organic Photochemistry and Photobiology, 2^(nd) ed. (eds.Horspool, W. & Lenci, F.) 89.1-89.16 (CRC Press, Boca Raton, Fla.,2004). Current block in the dark followed a bi-exponential time course,suggesting that a second process was involved. This may be a decrease inslow inactivation as the channels become re-blocked by QA. Choi, et al.Proc. Natl. Acad. Sci. USA 88, 5092-5095 (1991).

FIGS. 2A-D. Photocontrol of MAL-AZO-QA-modified Shaker channels inXenopus oocytes. (a) Raw Shaker K⁺ current traces recorded from anoutside-out patch before and after treatment with MAL-AZO-QA. Scalebars: 100 pA (vertical) and 50 msec (horizontal). The top trace in eachpanel shows the current before MAL-AZO-QA application. Bottom tracesrepresent current after 4 min application of 10 μM MAL-AZO-QA and 2 minwashout (left panel), after 1 min exposure to ultraviolet (380 nm) light(middle panel), and after 1 min exposure to visible (500 nm) light(right panel). The patch was held at −90 mV and currents were elicitedby 100 msec steps to −20 mV at 1 Hz. (b) K⁺ current amplitudes from thesame outside-out patch during perfusion with MAL-AZO-QA, during washout,and during alternating illumination with 380 and 500 nm light. (C)Inside-out patch from an oocyte treated with 100 μM MAL-AZO-QA for 30min. The patch shows a large Shaker current in 380 nm light and almostcomplete block in 500 nm light. Pulse protocol same as above, exceptpulse duration was 30 msec. (D) Current block in dark follows abiexponential timecourse with τ₁=0.49 min and τ₂=4.79 min.

Which wavelengths are best for opening and closing MAL-AZO-QA-modifiedchannels? To address this question, the absorbance spectra of MAL-AZO-QAwas measured in solution as a glutathione adduct (FIG. 3 a). The transconfiguration of MAL-AZO-QA exhibits a large absorbance peak at 360 nmand small shoulder at ˜440 nm, as reported previously for other AZOderivatives. Knoll, supra. Maximal photoisomerization to the cisconfiguration considerably decreased the 360 nm peak and slightlyincreased absorbance between 440 and 540 nm. Although the spectraindicate the wavelengths of maximum absorbance for the trans and cisisomers, the spectral overlap between isomers suggests that these maynot be the optimal wavelengths for maximal photoconversion. In addition,coupling of MAL-AZO-QA to the channel protein could affect theabsorbance spectra. The optimal wavelengths were determined empiricallyby measuring the action spectrum of each isomer (FIG. 3 b-d).

To determine the wavelength that results in maximal recovery of K⁺currents, inside-out patches were initially exposed to long wavelengthlight (500 nm) from a Xenon lamp for at least 1 min to maximizeoccupancy of the blocked state. The patch was then irradiated with adiscrete wavelength between 300-480 nm for 1 min, and the peak currentat steady-state was measured to determine the degree of unblock (FIG. 3b). The resulting action spectrum shows that 380 nm is most effective inunblocking MAL-AZO-QA-modified Shaker channels (FIG. 3 d. The actionspectrum for channel unblock should reflect the steady-state ratio oftrans and cis isomers (the photostationary state) at each wavelength.

To determine the complementary action spectrum for channel re-block,patches were exposed to 380 nm for 1 min to maximize occupancy of thecis state. Subsequent exposure to discrete wavelengths between 420 and600 nm caused re-block of the channels at different rates (FIG. 3 c). Inthis case, rate of re-block is the most relevant parameter, since manywavelengths will eventually result in complete block. Thus we measuredthe degree of block at a fixed time (0.2 min) for each wavelength. Thebroad peak of this action spectrum (FIG. 3 d) suggests that wavelengthsfrom 460 to 500 nm cause the fastest re-block of channels.

FIGS. 3A-D. Absorbance and action spectra of MAL-AZO-QA. (a) The UV/VISspectrum of a MAL-AZO-QA-glutathione adduct (10 μM) in oocyte bathsolution. To maximize the trans and cis isomers, the solution wasexposed to visible and ultraviolet light, respectively, for 3 minutes.To generate the adduct, MAL-AZO-QA (1 M) was treated with reducedglutathione (1.5 M) in for 12 hrs at 21° C. (b) Unblock of Shakerchannels at different wavelengths. Currents are from an inside-out patchalternately exposed to various wavelengths between 300-480 nm to unblockthe channels, and 500 nm light to re-block the channels. (c) Re-block ofShaker channels at different wavelengths. The timecourse of block atvarious wavelengths of visible light. Each trial is preceded by 1 minirradiation at 380 nm to unblock the channels. Traces are superimposedfor comparison. Normalized current amplitudes were measured at 0.2 minafter onset of block. (D) Action spectra for unblock (left curve) andblock (right curve) of Shaker K⁺ channels (n=3-8 patches for eachwavelength). Unblock (left axis): Current unblocked at each wavelengthdivided by current at 380 nm. Currents were compared within each patch.Block (right axis): Fraction of normalized current blocked at 0.2 minafter illumination with visible light (n=2-7 patches for eachwavelength).

It was determined whether light-activated channels could be used tocontrol neuronal excitability. First, the voltage-dependence of thechannel was modified, so that the photoswitch is the primary regulatorof gating. Normally, Shaker K⁺ channels make only a minor contributionto the membrane conductance at typical resting potentials (−40 to −70mV). The channels also display voltage-dependent inactivation, furtherlimiting their contribution. Mutations were therefore introduced toeliminate rapid inactivation (46-46; Hoshi, et al. Science 250, 533-538(1990)), reduce slow inactivation (T449V; Lopez-Barneo, et al. ReceptorsChannels 1, 61-71 (1993)), and shift voltage-dependent activation tohyperpolarized potentials (L366A; Lopez, et al. Neuron 7, 327-336(1991)), as confirmed by expression in oocytes. Expression ofvoltage-gated K⁺ channels with these modifications should result in ahigh resting K⁺ conductance and silencing of spontaneous activity.

This multiply-mutated Shaker channel was expressed in culturedhippocampal neurons, which were subsequently treated with MAL-AZO-QA for15 min in the dark, followed by thorough washout. Current clamprecordings from transfected pyramidal cells, identified by co-expressionof GFP, showed that exposure to 390 nm light silenced spontaneous actionpotentials within 3 sec and exposure to 500 nm light restored activityalso within seconds (FIG. 4 a). Similar results were obtained in 5cells. Activity could also be restored simply by leaving neurons in thedark after silencing, but the onset was slow (>30 sec), in accord withthe slow re-block of ionic current observed in oocyte patches in thedark. Hence a 5 sec pulse of 390 nm light should produce relativelysustained silencing. Prolonged depolarizing current steps causedrepetitive firing in 500 nm light (FIG. 4 b, left). In contrast, in 390nm light the same steps elicited rapidly accommodating responses(usually a single action potential, even with depolarization well abovethreshold) (FIG. 4 b, right). On average, 390 nm light decreased thenumber of action potentials elicited by a depolarizing step by 79% (n=4)(FIG. 4 c).

Light had no effect on MAL-AZO-QA-treated neurons that expressed GFPwithout the multiply-mutated Shaker channels (n=4), nor onchannel-transfected neurons without MAL-AZO-QA treatment (n=5). Hence,it seems that native K⁺ channels are not susceptible to light-regulatedblock by tethered MAL-AZO-QA, even though many of these channels can beblocked by TEA. The observation that only Shaker-transfected neurons arelight sensitive suggests that MAL-AZO-QA selectively attaches to theintroduced cysteine, which is facilitated by the high effective localconcentration of the cysteine-reactive maleimide when the quaternaryammonium binds to the pore. Blaustein, J. Gen. Physiol. 120, 203-216(2002). Non-selective attachment of MAL-AZO-QA to extracellularcysteines on other membrane proteins may have no detectable effects oncellular electrophysiology, since other channels and receptors areunlikely to have a TEA-binding site positioned at the appropriatedistance from a modifiable cysteine.

FIGS. 4A-C. Expression of light-activated channels conferslight-sensitivity on hippocampal pyramidal neurons. (a) Spontaneousaction potentials are silenced and revived by exposure to 390 and 500 nmlight respectively. The neuron, transfected with the multiply-mutatedShaker channel, was treated for 15 min with MAL-AZO-QA before recording.The frequency of spontaneous synaptic potentials generated byuntransfected neurons is not affected by light. (b) Depolarizing currentsteps elicit repetitive firing in 500 nm light (left) but only singleaction potentials in 390 nm light (right). Neurons were held undercurrent clamp at ˜−55 mV and depolarized up to ˜−15 mV. (c) Summary ofrepetitive firing data. Number of spikes resulting from a suprathresholddepolarization to −15 mV is significantly modulated by light in themultiply mutated Shaker-transfected neurons treated with MAL-AZO-QA (*:p<0.01). Neurons expressing the channel without MAL-AZO-QA treatment, ortreated with MAL-AZO-QA without channel expression, were unaffected bylight.

Example 2 Light-Regulated Ionotropic Glutamate Receptor (iGluR)

The iGluR family members mediate the major excitatory currents in thecentral nervous system⁸. Structurally, they are tetrameric proteinassemblies whose subunits consist of an extracellular N-terminal domain(NTD), an extracellular ligand binding domain (LBD), and a transmembranedomain (TMD) that forms the pore (FIG. 5 b)⁹. The LBD closes like aclamshell as it binds the agonist glutamate. This reversible binding andclosure is allosterically coupled, in an as yet unknown way, to theopening of the pore. The detailed structures of the LBD of severaliGluR's in their apo state or in complex with agonists (e.g. glutamate,kainate, AMPA or domoic acid) have been solved by X-raycrystallography¹⁰⁻¹². These structures provide a vivid picture of howthe LBD closes when the agonist binds.

The approach to engineering LiGluR was to site-specifically attach atethered analogue of glutamate containing a photoisomerizable azobenzenemoiety to a “lip” of the LBD clamshell (FIG. 5 c). In one state of theazobenzene, the LBD would not bind the tethered agonist and thereforeremain open. Only after isomerization would the tether present theagonist to the binding site and thus effect closure. Overall, thereversible switching of an azobenzene would allosterically trigger theopening and closing of the entire ion channel, mediated by theclamshell-like movement of the LBD.

FIGS. 5A-C Design of an allosteric photoswitch. (a) An agonist (orange)is tethered to a LBD through an optical switch (red) via linkers(black). In one state of the switch, the ligand cannot reach the bindingpocket, whereas in the other state the ligand docks and stabilizes theactivated (closed) conformation of the LBD. (b) Schematic representationof the operating mode of iGluRs. Binding of an agonist (orange)stabilizes the activated (closed) conformation of the LBD andallosterically opens the pore, allowing flow of Na⁺, Ca²⁺ andNBD=N-terminal domain. TMD=transmembrane domain. (c) The principle ofLiGluR. Reversible optical switching of a tethered agonist on the LBDopens and closes the pore.

Methods

Synthetic Protocols.

General Information

All non-aqueous reactions were performed using flame- or oven-driedglassware under an atmosphere of dry nitrogen. Commercial reagents wereused as received. Non-aqueous reagents were transferred under nitrogenwith a syringe or cannula. Solutions were concentrated in vacuo on aBuchi rotary evaporator. Diisopropylethylamine (DIPEA) was distilledfrom calcium hydride prior to use. Tetrahydrofuran (THF) and methylenechloride (CH₂Cl₂) were passed through a column of activated aluminaunder N₂-pressure prior to use. N,N-Dimethyl formamide (DMF) wasdegassed with a stream of N₂, dried over molecular sieves, and usedwithout further purification. Chromatographic purification of productswas accomplished using flash column chromatography on ICN 60 32-64 meshsilica gel 63 (normal phase) or Waters Preparative C18 125 Å 55-105 μmsilica gel (reversed phase), as indicated. Thin layer chromatography(TLC) was performed on EM Reagents 0.25 mm silica gel 60-F₂₅₄ plates.Visualization of the developed chromatogram was performed usingfluorescence quenching, KMnO₄, ceric ammonium molybdate (CAM), or iodinestains. IR spectra were measured with a Genesis FT-IR spectrometer bythin film or Avatar 370 FT-IR by attenuated total reflectance accessory.Optical rotations were measured using a Perkin-Elmer 241 Polarimeter at25° C. and 589 nm. ¹H and ¹³C NMR spectra were recorded in deuteratedsolvents on Bruker AVB-400, AVQ-400, or DRX-500 spectrometers andcalibrated to the residual solvent peak. Multiplicities are abbreviatedas follows: s=singlet, d=doublet, t=triplet, q=quartet, m=multiplet,app=apparent, br=broad.

A solution of 2 (321 mg, 1.07 mmol) and freshly distilled acrylic acid(231 mg, 3.21 mmol) in CH₂Cl₂ (5.0 mL) was added directly to solidGrubbs' 2^(nd) generation catalyst (43 mg, 0.025 mmol). The mixture washeated to reflux for 12 h. The reaction mixture was then concentratedand purified by normal phase chromatography (8.5:1.5 CH₂Cl₂:EtOAc with1% AcOH) to yield S4 (317 mg, 92%) as a tan oil. Data for S4: R_(f) 0.15(5:4 hexanes:EtOAc with 1% AcOH); [α]_(D)=−21.8 (c 1.0, in CH₂Cl₂); IR:2981, 2937, 1788, 1742, 1717, 1653 cm⁻¹; ¹H NMR (CDCl₃, 400 MHz) δ 6.98(m, 1H), 5.90 (d, 1H, J=15 Hz), 4.57 (d, 1H, J=9 Hz), 4.24 (q, 2H, J=7Hz), 2.82 (m, 2H), 2.34 (m, 1H), 2.25 (m, 1H), 1.97 (m, 1H), 1.51 (s,9H), 1.27 (t, 3H, J=7 Hz); ¹³C NMR (CDCl₃, 100 MHz) δ 173.4, 171.0,149.2, 147.2, 123.3, 83.8, 61.8, 57.0, 40.6, 32.8, 28.1, 27.8, 20.7,14.1; LRMS (ESI) Calc for C₁₆H₂₂NO₇ (M-H)⁻: 340.1. Found: 340.1.

To a solution of S4 (80.0 mg, 0.234 mmol) in MeOH (10.0 mL) was addedPd/C (25 mg, 0.023 mmol). The resulting suspension was stirred at roomtemperature under a hydrogen atmosphere for 12 h. The suspension wasthen filtered through celite and concentrated to yield 9 (78 mg, 97%) asa tan oil. Data for 9: R_(f) 0.15 (5:4 hexanes:EtOAc with 1% AcOH);[α]_(D)=−18.9 (c 1.0, in CH₂Cl₂); IR: 2980, 2936, 1787, 1742, 1719 cm⁻¹;¹H NMR (CDCl₃, 400 MHz) δ 4.55 (d, 1H, J=9 Hz), 4.24 (q, 2H, J=7 Hz),2.61 (m, 1H), 2.38 (m, 2H), 2.24 (m, 1H), 1.95 (m, 2H), 1.68 (m, 2H),1.48 (s, 9H), 1.42 (m, 1H), 1.28 (t, 3H, J=7 Hz); ¹³C NMR (CDCl₃, 100MHz) δ 178.7, 174.7, 171.2, 149.4, 83.5, 61.7, 57.1, 41.4, 33.6, 29.7,28.4, 27.8, 21.9, 14.1; LRMS (ESI)⁻ Calc for C₁₆H₂₄NO₇ (M-H)⁻: 342.2.Found: 342.2.

To a solution of S2 (83.7 mg, 0.316 mmol), 1-Hydroxy-benzotriazolehydrate (HOBt) (64.3 mg, 0.475 mmol), DIPEA (441 μL, 2.53 mmol), andN-Ethyl-N′-(3-dimethyldiaminopropyl)-carbodiimide.HCl (EDCI) (79.0 mg,0.412 mmol) in CH₂Cl₂ (12.0 mL) was added a solution of 9 (130 mg, 0.380mmol) in CH₂Cl₂ (5.0 mL). The mixture was stirred at room temperaturefor 12 h. The mixture was diluted with CH₂Cl₂ (60 mL) and washed with asaturated NaHCO₃ solution (2×100 mL) and brine (2×100 mL). The organiclayer was dried over Na₂SO₄, filtered, and concentrated. Purification bynormal phase chromatography (95:5 CH₂Cl₂:MeOH) gave S3 (97 mg, 65%) as awhite solid. Data for S3: R_(f) 0.29 (95:5 CH₂Cl₂: MeOH); mp 57-59° C.;[α]_(D)=−14.4 (c 1.0, in CH₂Cl₂); IR: 3324, 2980, 2936, 2359, 2251,1784, 1744, 1716, 1653, 1601, 1547 cm⁻¹; ¹H NMR (CDCl₃, 500 MHz) δ 8.83(s, 1H), 7.50 (d, 2H, J=8 Hz), 7.28 (t, 2H, J=8 Hz), 7.06 (t, 2H, J=7Hz), 4.87 (d, 1H, 9 Hz), 4.20 (q, 2H, J=7 Hz), 4.08 (t, 2H, J=5 Hz),2.59 (m, 1H), 2.31 (m, 2H), 2.18 (m, 1H), 1.89 (m, 2H), 1.70 (m, 2H),1.44 (s, 9H), 1.39 (m, 1H), 1.26 (t, 3H, J=7 Hz); ¹³C NMR (CDCl₃, 125MHz) δ 175.3, 173.7, 171.2, 167.5, 149.3, 137.8, 128.9, 124.3, 120.0,83.7, 61.7, 57.2, 44.4, 41.4, 35.7, 29.7, 28.3, 27.8, 22.8, 14.2; HRMS(FAB) Calc for C₂₄H₃₃N₃O₇ (M)⁺: 475.231851. Found: 475.232400.

To a solution of S3 (96.0 mg, 0.200 mmol) in THF (2.0 mL) was added a1.0 M aqueous solution of LiOH (2.0 mL). The mixture was stirred at 0°C. for 1 h and then acidified to pH 2 with a 1.0 M HCl solution andextracted with EtOAc (3×20 mL). The combined organic layers were driedover Na₂SO₄, filtered, and concentrated to give an oily residue that wasreacted with a saturated HCl solution in EtOAc for 2 h at roomtemperature. The resulting white solid was triturated with ethyl ether(3×20 mL) to yield 3 (68.5 mg, 85%). Data for 3: mp 154-156° C.;[α]_(D)=−11.4 (c 0.7, in H₂O); IR: 2933, 1709, 1598, 1544 cm⁻¹; ¹H NMR(D₂O, 500 MHz) δ 7.30 (m, 4H), 7.13 (m, 1H), 3.92 (s, 2H), 3.87 (m, 1H),2.58 (m, 1H), 2.26 (m, 2H), 2.21 (m, 1H), 1.85 (m, 1H), 1.55 (m, 4H);¹³C NMR (DMSO-d₆, 125 MHz) δ 175.6, 172.4, 170.8, 167.9, 139.0, 128.7,123.1, 119.1, 50.8, 42.7, 40.4, 35.0, 34.2, 31.2, 22.5; LRMS (ESI)⁻ Calcfor C₁₇H₂₂N₃O₆ (M-H)⁻: 364.2. Found: 364.1.

To a solution of azodianiline 5 (750 mg, 3.50 mmol), HOBt (135 mg, 5.25mmol), DIPEA (2.40 mL, 14.0 mmol), and EDCI (872 mg, 4.55 mmol) inCH₂Cl₂ (100 mL) was added a solution of Boc-Gly-OH (674.0 mg, 3.85 mmol)in CH₂Cl₂ (20 mL). The mixture was stirred at room temperature for 12 h.The mixture was diluted with CH₂Cl₂ (400 mL) and washed with a saturatedNaHCO₃ solution (2×400 mL) and brine (2×400 mL). The organic layer wasdried over Na₂SO₄, filtered, and concentrated. Purification by normalphase chromatography (dry loaded, 10:0→7:3 CH₂Cl₂:EtOAc) gave S5 (581mg, 66%) as an orange solid. Data for S5: R_(f) 0.21 (95:5 CH₂Cl₂:MeOH); mp 184-185° C.; UV λ_(max) (MeOH): 391 nm; IR: 3306, 2424, 1705,1673, 1602, 1531, 1501 cm⁻¹; ¹H NMR (MeOH-d₄, 400 MHz) δ 7.75 (d, 2H,J=9 Hz), 7.68 (d, 4H, J=9 Hz), 6.72 (d, 2H, J=9 Hz), 3.88 (s, 2H), 1.46(s, 9H); ¹³C NMR (DMSO-d₆, 125 MHz) δ 168.5, 156.0, 152.4, 148.0, 142.8,140.2, 124.8, 122.5, 119.3, 113.4, 78.1, 43.9, 28.2; HRMS (FAB) Calc forC₁₉H₂₄N₅O₃ (MH)⁺: 370.187915. Found: 370.187140.

To a solution of S5 (565 mg, 1.53 mmol) in a 9:1 mixture of CH₂Cl₂:MeOH(50 mL) was added trifluoroacetic acid (50 mL). The mixture was stirredfor 4 h at room temperature, concentrated, and triturated with diethylether (2×100 mL) to yield 6 (695 mg, 98%) as a purple solid. Data for 6:mp>180° C. dec; UV λ_(max) (MeOH): 394 nm; IR: 2879, 2637, 1673, 1621,1601, 1542, 1502 cm⁻¹; ¹H NMR (MeOH-d₄, 400 MHz) δ 7.81 (d, 2H, J=9 Hz),7.73 (m, 4H), 6.84 (d, 2H, J=9 Hz), 3.89 (s, 2H); ¹³C NMR (DMSO-d₆, 125MHz) δ 165.6, 150.4, 149.5, 147.3, 140.9, 126.1, 124.1, 121.1, 117.5,42.3; HRMS (FAB) Calc for C₁₄H₁₆N₅O (MH)⁺: 270.135485. Found:270.135930.

To a solution of 6 (180 mg, 0.387 mmol), HOBt (78.6 mg, 0.581 mmol),DIPEA (269 μL, 1.55 mmol), and EDCI (96.4 mg, 0.503 mmol) in CH₂Cl₂ (20mL) was added a solution of 9 (159 mg, 0.465 mmol) in CH₂Cl₂ (5.0 mL).The mixture was stirred at room temperature for 12 h. The mixture wasdiluted with CH₂Cl₂ (250 mL) and washed with a saturated NaHCO₃ solution(2×200 mL) and brine (2×200 mL). The organic layer was dried overNa₂SO₄, filtered, and concentrated. Purification by normal phasechromatography (97:3→95:5 CH₂Cl₂:MeOH) gave S6 (223 mg, 97%) as anorange solid. Data for S6: R_(f) 0.32 (95:5 CH₂Cl₂: MeOH); mp 115-117°C.; [α]_(D)=−9.8 (c 1.0, in CH₂Cl₂); UV λ_(max) (CHCl₃): 382 nm; IR:3358, 2361, 1781, 1741, 1697, 1651, 1598, 1540 cm⁻¹; ¹H NMR (CDCl₃, 400MHz) δ 8.97 (s, 1H), 7.80 (d, 2H, J=9 Hz), 7.76 (d, 2H, J=9 Hz), 7.66(d, 2H, J=9 Hz), 7.03 (m, 1H), 6.72 (d, 2H, J=9 Hz), 4.52 (dd, 1H, J=9Hz), 4.21 (q, 2H, J=7 Hz), 4.12 (m, 2H), 4.07 (s-br, 2H), 2.67 (m, 1H),2.33 (s, 2H), 2.26 (m, 1H), 1.94 (m, 2H), 1.74 (m, 3H), 1.46 (s, 9H),1.27 (t, 3H, J=7 Hz); ¹³C NMR (CDCl₃, 100 MHz) δ 175.6, 173.7, 171.1,167.6, 149.6, 149.2, 145.2, 139.3, 124.8, 123.1, 119.9, 114.5, 83.7,61.7, 57.2, 44.3, 41.3, 35.6, 29.7, 28.1, 27.7, 22.7, 14.1; HRMS (FAB)Calc for C₃₀H₃₈N₆O₇ (M)⁺: 594.280198. Found: 594.280490.

To a solution of Fmoc-Gly-OH (600 mg, 2.02 mmol) and oxalyl chloride(1.2 mL of 2.0 M solution in THF, 2.4 mmol) in CH₂Cl₂ (6.0 mL) was addedone drop of DMF. After stirring for 1 h at room temperature the mixturewas concentrated. The resulting acid chloride was redissolved in THF(11.0 mL) and added via cannula to a solution of S6 (400 mg, 0.672mmol), DIPEA (586 μL, 3.36 mmol), and 4-(Dimethylamino)-pyridine (DMAP)(8.2 mg, 0.067 mmol) in THF (26.0 mL). After stirring 10 min at 0° C.,the mixture was warmed to room temperature and stirred an additional 3h. The mixture was then diluted with CH₂Cl₂ (150 mL) and washed with asaturated solution of NaHCO₃ (2×150 mL) and brine (2×150 mL). Theorganic layer was dried over Na₂SO₄, filtered, and concentrated.Purification by normal phase chromatography (90:10:0.6:0.6CH₂Cl₂:MeOH:AcOH:H₂O) gave the Fmoc-glycine adduct as an orange solidthat was sufficiently pure for further reaction. To a solution of thiscompound in DMF (20.0 mL) was added piperidine (200 μL, 2.02 mmol).After stirring for 6 h at room temperature, the mixture was concentratedand purified by reversed phase chromatography (4:1→2:3 0.1% formic acidin H₂O:MeCN) to yield 10 (186 mg, 43%) as an orange solid. Data for 10:mp>210° C. dec; [α]_(D)=−15.5 (c 0.3, in MeOH); UV λ_(max) (MeOH): 365nm; IR: 3280, 2981, 1781, 1690, 1593, 1546 cm⁻¹; ¹H NMR (MeOH-d₄, 500MHz) δ 7.88 (m, 4H), 7.76 (m, 4H), 4.63 (d, 1H, J=9 Hz), 4.24 (m, 2H),4.03 (s, 2H), 3.85 (s, 2H), 2.64 (m, 1H), 2.34 (m, 2H), 2.28 (m, 1H),2.08 (m, 1H), 1.89 (m, 1H), 1.72 (m, 2H), 1.47 (s, 9H), 1.42 (m, 1H),1.29 (t, 3H, J=7 Hz); ¹³C NMR (MeOH-d₄, 100 MHz) δ 178.0, 176.5, 173.2,170.1, 169.5, 151.0, 150.6, 150.3, 142.6, 141.9, 124.8, 124.7, 121.3,121.1, 84.9, 63.0, 59.0, 45.9, 44.3, 42.8, 36.6, 31.1, 29.2, 28.3, 24.1,14.7; HRMS (FAB) Calc for C₃₂H₄₁N₇O₈ (M)⁺: 651.301662. Found:651.304080.

To a solution of 10 (180 mg, 0.276 mmol) in THF (10.0 mL) at 0° C. wasadded 1.0 M LiOH (10.0 mL). After stirring for 1 h, the mixture wasacidified to pH 2 with 1 M HCl, THF was removed in vacuo, and themixture was purified by reversed phase chromatography (5:0→4:1 0.1%formic acid in H₂O:MeCN) to yield S7 (143 mg, 80%) as a yellow solid.Data for S7: mp>230° C. dec; [α]_(D)=−15.9 (c 0.1, in DMSO); UV λ_(max)(DMSO): 379 nm; IR: 2981, 1690, 1660, 1595, 1546, 1500 cm⁻¹; ¹H NMR(MeOH-d₄, 500 MHz) δ 7.87 (m, 4H), 7.76 (m, 4H), 4.07 (m, 1H), 4.03 (d,2H, J=5 Hz), 3.88 (s, 2H), 2.55 (m, 1H), 2.32 (m, 2H), 2.20 (m, 1H),1.70 (m, 4H), 1.59 (m, 1H), 1.42 (s, 9H); ¹³C NMR (DMSO-d₆, 125 MHz) δ176.4, 174.2, 172.5, 168.5, 163.8, 155.4, 147.8, 147.6, 141.7, 141.2,123.5, 123.4, 119.3, 79.2, 77.9, 52.5, 44.0, 42.9, 41.6, 35.0, 32.0,28.2, 23.0; LRMS (ESI)⁻ Calc for C₃₀H₃₈N₇O₉ (M-H)⁻: 640.3. Found: 640.2.

To a solution of S7 (53 mg, 0.082 mmol) in a saturated solution ofNaHCO₃ (3.5 mL) was added finely ground N-methoxycarbonylmaleimide (105mg, 0.677 mmol) under vigorous stirring. After 30 min at 0° C., themixture was diluted with THF (3.5 mL) and warmed to room temperature.After 1 h, the mixture was acidified to pH 1-2 with an aqueous solutionof 1.0 M H₂SO₄ and extracted with EtOAc (2×20 mL). The combined organiclayers were dried over Na₂SO₄, filtered, and concentrated. Purificationby normal phase chromatography (90:10:0.6:0.6 CH₂Cl₂:MeOH:AcOH:H₂O) gaveS8 (42 mg, 71%) as a yellow solid. Data for S8: R_(f) 0.20(90:10:0.6:0.6 CH₂Cl₂:MeOH:AcOH:H₂O); mp>230° C. dec; [α]_(D)=−14.0 (c0.1, in MeOH); UV λ_(max) (MeOH): 365 nm; IR: 3249, 3186, 2928, 1753,1708, 1687, 1651, 1520 cm⁻¹; ¹H NMR (DMSO-d₆, 500 MHz) δ 10.61 (s, 1H),10.31 (s, 1H), 8.19 (m, 1H), 7.84 (d, 4H, J=9 Hz), 7.79 (d, 2H, J=9 Hz),7.75 (d, 2H, J=9 Hz), 7.16 (s, 2H), 7.10 (d, 2H, J=8 Hz), 4.32 (s, 2H),3.82 (m, 1H), 3.64 (s, 2H), 2.35 (m, 1H), 2.15 (m, 2H), 1.93 (m, 1H),1.59 (m, 1H), 1.49 (m, 3H), 1.42 (m, 1H), 1.36 (s, 9H); ¹³C NMR(DMSO-d₆, 125 MHz) δ 176.1, 174.2, 172.5, 170.7, 168.4, 166.6, 155.7,147.9, 147.6, 141.7, 141.1, 135.0, 123.5, 123.4, 119.5, 119.3, 78.0,52.4, 42.8, 41.4, 40.5, 45.0, 32.8, 32.1, 28.2, 22.9; LRMS (ESI)⁻ Calcfor C₃₄H₃₈N₇O₁₁ (M-H)⁻: 720.3. Found: 720.3.

To a flask containing solid S8 (38 mg, 0.053 mmol) was added a saturatedHCl solution in EtOAc (25.0 mL). After stirring at room temperature for2 h, the resulting purple solid was triturated with ethyl ether (2×40mL) to yield 4 (30 mg, 87%). Data for 4: mp>230° C. dec; =[α]_(D)19.5 (c0.7, in DMSO); UV λ_(max) (10% DMSO in H₂O): 363 nm; IR: 3279, 3052,2935, 2362, 1922, 1709, 1598, 1538 cm⁻¹; ¹H NMR (DMSO-d₆, 500 MHz) δ10.70 (s, 1H), 10.39 (s, 1H), 8.23 (m, 1H), 7.84 (d, 4H, J=8 Hz), 7.79(d, 2H, J=9 Hz), 7.76 (d, 2H, J=9 Hz), 7.16 (s, 2H), 4.32 (s, 2H), 3.91(d, 2H, J=6 Hz), 3.81 (m, 1H), 2.89 (m, 1H), 2.16 (m, 2H), 2.13 (m, 1H),2.77 (m, 1H), 1.53 (m, 4H); ¹³C NMR (DMSO-d₆, 125 MHz) δ 175.5, 172.4,170.9, 170.7, 168.4, 165.3, 147.8, 147.6, 141.7, 141.1, 135.0, 123.5,123.4, 119.4, 119.3, 64.9, 50.8, 42.7, 34.9, 31.7, 31.2, 22.4, 15.2;HRMS (ESI)⁺ Calc for C₂₉H₃₂N₇O₉ (MH)⁺: 622.2262. Found: 622.2268.

Site Directed Mutagenesis

Cysteine point mutations were introduced to the iGluR6 DNA, containing Qat the position 621 RNA editing site¹⁵ using the QuikChangesite-directed mutagenesis kit (Stratagene). The following PCR profilewas used: one cycle (95° C. for 30 s); 20 cycles (95° C. for 30 s, 55°C. for 1 min, 68° C. for 12 min). The forward and reverseoligonucleotide sequences designed for the L439C mutant were:

(SEQ ID NO: 1) 5′-GATTGTTACCACCATTTGCGAAGAACCGTATGTTCTG-3′; and(SEQ ID NO: 2) 5′-CAGAACATACGGTTCTTCGCAAAATGGTGGTAACAATC-3′.Cell Culture and Transfection

HEK293 cells were plated at approximately 3×10⁶ cells/ml onpoly-1-lysine-coated glass coverslips (Deutsche Spiegelglas, CarolinaBiological) and maintained in DMEM with 5% fetal bovine serum, 0.2 mg/mlstreptomycin, and 200 U/ml penicillin at 37° C. Cells were transientlytransfected with various plasmids using lipofectamine 2000 (Invitrogen).The amount of total transfected iGluR6 DNA and enhanced yellowfluorescent protein (EYFP) fusion DNA per well was fixed at 4 μg and 200ng, respectively. All recordings were carried out 36 to 48 h aftertransfection.

Attachment of MAG

To conjugate MAG to cysteine mutants of iGluR6, the compound was dilutedto 10-100 μM (final concentration 0.5-5% DMSO) in the HEK cell controlsolution and the cells were incubated in the dark for 15-30 min.

Calcium Imaging

Cells were washed in PBS and loaded with 5 μM FURA-2-AM (MolecularProbes) for 30 min. Changes of [Ca²⁺], in individual cells were measuredas intracellular Fura2 fluorescence intensity using mercury arc lampillumination and alternating excitation with band pass filters of 350 nmand 380 nm during 66 ms at 5-20 s intervals and detecting emission at510 nm¹⁶. Fluorescence was monitored on an inverted microscope system(Nikon). Images were captured with a CCD camera using the ImagingWorkbench software, which was also used to irradiate the cells at 380and 500 nm during 1-2 min in order to produce photoisomerization of MAG.Measurements were performed in a control solution (in mM): 135 NaCl, 5.4KCl, 0.9 MgCl₂, 1.8 CaCl₂, HEPES, 10 glucose, and pH 7.6, containing 300mg/l concanavalin A type IV (Sigma) to block desensitization^(29,30).L-Glutamate was applied as reported in text and figures. The results arerepresentative data from multiple cells in at least two independentcultures.

Whole-Cell Patch Clamping

Patch clamp recordings were carried out using an Axopatch 200A amplifierin the whole cell mode. Cell voltage was held at −60 mV. Pipettes hadresistances 4-8 MΩ and were filled with a solution containing (in mM):145 CsCl, 5 EGTA, 0.5 CaCl₂, 1.0 MgCl₂, 10 HEPES, pH7.2. Illuminationwas applied using a TILL Photonics Polychrome II monochromator through a60×/1.2 W objective (power output: 12.4 W/m² irradiance; 500 nm asmeasured with a Newport optical power meter). Data was recorded withpClamp software, which was also used to control the monochromator.

Results

The design of a tethered agonist was based on extensivestructure-activity relationship analyses that have been performed oniGluR agonists^(13,14) and, importantly, on the X-ray structure of theLBD of iGluR6 in complex with the agonist (2S,4R)-4-methyl glutamate (1)(FIG. 6 c)¹¹. From this structure, it can be seen that the ligand-boundform of the clamshell, although closed, features a narrow “exitchannel.” It was believed that the exit channel would enable a tetherappended to an agonist to protrude and reach an attachment site at thesurface of the protein, while still permitting the clamshell to closeover the agonist and activate.

To explore the feasibility of this idea, a “tether model”, termedMV-2-025 (3) (FIG. 6 a), was synthesized. This compound is in essence analkylated version of glutamate and resembles the known iGluR6 agonist(2S,4R)-4-allyl glutamate (2) (FIG. 6 a)¹⁴. The allyl side chain of thiscompound was extended to include a moiety that mimics half of anazobenzene. This partial tether should, in principle, be long enough toproject out of the exit channel and thus serve as a reasonable basis fordetermining if a full-length azobenzene tether would impede LBDactivation.

FIGS. 6A-D. Structures and fit of photoswitched agonist and iGluR6 LBD.(a) Chemical structure of the iGluR6 agonists (2S,4R)-4-methyl glutamate1, (2S,4R)-4-allyl glutamate 2 and tether model 3. (b) Structure of MAG4 in its trans state (dark and 500 nm) and cis state (380 nm). (c) Viewlooking into the “mouth” of iGluR6 LBD in complex with 1 (PDB ID 1SD3)(10). Residues on clamshell “lips” that were individually mutated tocysteine are highlighted in yellow. Position 439 is shown in red. Themethyl group of 1 can be seen in blue at the bottom of the “exitchannel.” (d) Docking model of MAG in the cis state attached at L439C(yellow) and bound to the activated (closed) conformation of the LBD.

To assay LBD activation, the calcium permeability of iGluR6¹⁵ wasutilized. The iGluR6 was expressed in HEK293 cells, loaded the cellswith the fluorescent calcium indicator FURA-2-AM¹⁶, and the cells wereexposed to various concentrations of agonist 2, or of the tether model3, to quantify receptor activation (FIGS. 7A-E). Tether model 3 evokedlarge responses (FIG. 7 b). Allyl glutamate 2 had an EC₅₀ of 18 μM,while model 3 showed an EC₅₀ of 180 μM (FIG. 7 d). The maximal responseof tether model 3 was similar to that evoked by saturating glutamate,but ˜30% lower than that of the agonist 2 (FIG. 7 d), indicating thatthe side chain may interfere with clamshell closure to a minor degree.These results suggest that tethering a glutamate analogue is possiblewhile maintaining effective agonism. The loss in apparent affinity dueto the side chain of model 3 should be compensated for by the higheffective local concentration of a tethered ligand on its short leash.

FIGS. 7A-E Calcium imaging of iGluR6 activity. (a) Superimposed brightfield (grey) and EYFP (green) images of HEK293 cells co-transfected withiGluR6 and EYFP. (b) Calcium image (350/380 nm) of FURA-2-AM loadedcells during perfusion of 300 μM glutamate. Red and blue corresponds tohigh and low Ca²⁺ concentration, respectively. (c) Simultaneous Ca²⁺concentration traces from individual cells in response to indicatedconcentrations of the tether model 3. (d) Dose-response curves from Ca²⁺traces as in panel C. Higher concentrations of 3 activate iGluR6 atsimilar levels to saturating (1 mM) glutamate, to which responses arenormalized. Tether model 3 has an EC50 of 180 μM, compared to the higheraffinity molecule 2 on which it was based, which has an EC50 of 18 μM.(e) MAG 4 confers light sensitivity on iGluR6-L439C expressing cells(reversible increases in Ca²⁺ at 380 nm and decreases at 500 nm) but noton wildtype iGluR6 (WT). Agonism by free MAG is transient and reversesupon washout for both iGluR6-L439C and WT. IGluR6-L439C retains abilityto be activated by free glutamate after MAG 4 conjugation. Note thatCa²⁺ concentration is not measured during irradiation at 380 or 500 nm,or during conjugation.

After evaluation of the stereochemistry and synthetic accessibility ofseveral candidates, attention was focused on the tethered agonistcompound designated MAG (4) (FIG. 6 b). This compound features acysteine reactive maleimide (M), an azobenzene photoswitch (A), and aglutamate head-group (G). Since the iGluR X-ray structure, on which thedesign was based, only provides a snapshot of a flexible protein, acertain amount of conformational flexibility was also built into MAG 4by adding to the linker freely rotatable bonds. The UV-VIS spectra ofthe cis- and trans-isomers of soluble MAG 4 are typical of azobenzenes.MAG 4 was prepared by multi-step synthesis, featuring a Grubbs olefinmetathesis, several amide couplings and an intricate sequence ofprotective group manipulations as shown in Scheme 1, depicted in FIG. 8.The tether model 3 was prepared along similar lines.

In parallel to the synthetic work described herein, a series of singlecysteine mutants of iGluR6 was prepared by site-directed mutagenesis.The positions were chosen to form a perimeter around the exit channel,close to where the maleimide end of the tether was predicted to stickout (FIG. 6 c). Ca²⁺ imaging was used to search for cysteine mutantsthat would provide optical activation after covalent attachment of MAG4. Although Ca²⁺ imaging has slow kinetics and illumination atwavelengths that are absorbed by azobenzene, this assay enabled rapidtesting of attachment positions. Three were found that demonstratedclear responses to light in which Ca²⁺-concentration increased at 380 nmand declined back to basal levels at 500 nm. Of the three, the versionof the receptor with a cysteine at 439 (iGluR-L439C) had the largestresponses (FIG. 7 e). Because the rise in free cytoplasmic Ca²⁺concentration depends not only on influx through iGluRs, but also onCa²⁺ buffering and pumping, we turned for further characterization towhole cell patch clamping to directly measure the kinetics of channelgating and to obtain quantitative measures of activation efficiency.

FIG. 8. Scheme 1

Total Synthesis of MAG 4. Reagents and conditions: (a) Boc-Gly-OH, EDC,HOBt, DIPEA (66%); (b) TFA, CH₂Cl₂ (98%); (c) see ref. 15; (d) AcrylicAcid, 5% Grubbs' 2¹¹″ generation catalyst (92%); (e) H₂, Pd/C, MeOH(97%); (f) 6, EDCI, HOBt, DIPEA (97%); (g) Fmoc-Gly-OH, (COCl)₂, DMF;(h) Piperidine, DMF (43%, over two steps); (i) 1.0 M LiOH H₂O/THF, 0° C.(80%); (j) N-Methoxycarbonylmaleimide, NaHCO₃, THF/H₂O (71%); (k) HClsat'd EtOAc (87%). Boc, t-butoxycarbonyl; CH₂Cl₂, dichloromethane;(COCl)₂, oxalyl chloride; DIPEA, diisopropylethylamine; DMF,N,N-dimethylformamide; EDCI,N-ethyl-N′-(3-dimethyldiaminopropyl)-carbodiimide.HCl; EtOAc, ethylacetate; Fmoc, 9-fluorenylmehoxycarbonyl; Gly, glycine; HOBt,1-hydroxybenzotriazole hydrate; MeOH, methanol; Pd/C, palladium oncarbon; TFA, trifluoroacetic acid; THF, tetrahydrofuran.

As shown in FIGS. 9A and 9B, iGluR-L439C conjugated with MAG 4 (LiGluR)can be activated both by free glutamate and by illumination. Thephotostationary cis/trans ratio of azobenzenes depends on thewavelength, with maximum cis-state occupancy typically observed at ˜380nm and maximum trans-state occupancy observed at ˜500 nm^(1,18). Theconjugate was illuminated at 500 nm (to favor the inactive trans form)and illumination at wavelengths that ranged from 280 to 460 nm (tophotoisomerize to the active cis form) was tested. The shortest testwavelengths evoked no response, the intermediate wavelengths evokedsubstantial inward currents, and the longer wavelengths had smallerresponses. The largest current was at 380 nm, agreeing with peakphotoisomerization of free azobenzene to the cis form. To examine theopposite transition, the receptor was maximally activated with 380 nmillumination and tested wavelengths between 400 and 600 nm. Receptorswere most efficiently turned off at 500 nm, agreeing with the peakphotoisomerization of free azobenzene to the trans form.

Importantly, the photocurrents were fully reversible and highlyreproducible. Repeated switching between 380 and 500 nm evoked responsesof similar amplitude over a period of more than 30 minutes, consistentwith the resistance of azobenzenes to bleaching and demonstrating thatthe system is robust. Even with weak illumination from a standardfluorescence lamp, attenuated by passage through a monochromator andfiber guide, the receptor turned on and off rapidly (τ_(on-380 nm)=115±3ms and τ_(off-500 nm)=92.3±0.3 ms; mean±SEM, N=3) at a power of 12.4W/m² (irradiance at 500 nm).

LiGluR can be turned on and off with light, but also preserves theability to be activated by freely diffusible glutamate (FIGS. 9A, 9B).The currents generated by irradiation are smaller than currents evokedby saturating (300 μM) glutamate and by saturating (≧1 mM) tether model3. This could be due to incomplete labeling, however, we consider thatunlikely since increased exposure (in either concentration or time) toMAG 4 during the conjugation period did not change the size of theoptical response. Alternatively, MAG 4 may only permit partial closureof the LBD. Incomplete closure of the ligand-binding domain has beenpreviously linked to partial agonism in the related iGluR2 channel¹⁹.

FIGS. 9A and 9B Whole-cell patch-clamp current recordings from HEK293cells expressing iGluR6-L439C after conjugation of MAG 4. (a) Inwardcurrents (downward deflections, carried mainly by Na⁺ influx) inresponse to glutamate are preserved in LiGluR. Irradiation with shortwavelengths of light (280 to 480 nm, in 20 nm increments) shows maximalactivation at 380 nm. Irradiation with long wavelengths of light (400 to600 nm, in 20 nm increments) shows maximal deactivation at 500 nm.Alternation between 380 and 500 nm illumination evokes highlyreproducible responses. (b) Patch-clamp traces comparing responses ofLiGluR to saturating glutamate, optical switching and the titration ofthe tether model 3. Saturating responses elicited by 3 are slightlyhigher than by 380 nm irradiation, suggesting geometric constraints thatprevent the LBD from fully closing on MAG.

The efficient activation of iGluR6-L439C by MAG 4 can be explained by amodel that shows cis-MAG docked into the glutamate-binding site of theclosed (activated) conformation of the LBD (FIG. 6 d). It is apparentthat the linker between the glutamate head-group and the azobenzenemoiety can comfortably protrude through the exit channel, with theazobenzene almost completely exposed to solvent.

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Example 3 Light-Controlled Ionotropic Glutamate Receptor; AffinityLabeling

The analysis of cell signaling requires the rapid and selectivemanipulation of protein function. Such control has been elusive becausehigh ligand specificity usually derives from high affinity, yieldingslow unbinding kinetics. Moreover, drug action is difficult to controlspatially. Photoswitches were synthesized that covalently modify targetproteins and reversibly present and withdraw a ligand from its bindingsite due to photoisomerization of an azobenzene linker. The propertiesof a glutamate photoswitch that controls an ion channel in cells aredescribed here. Affinity labeling and geometric constraints ensure thatthe photoswitch controls only the targeted channel. Photoswitching tothe activating state places a tethered glutamate at a high (millimolar)effective local concentration near the binding site. The fraction ofactive channels can be set in an analog manner by altering thephotostationary state with different wavelengths. The bi-stablephotoswitch can be turned on with millisecond long pulses at onewavelength, remain on in the dark for minutes, and turned off withmillisecond long pulses at the other wavelength, yielding sustainedactivation with minimal irradiation. The system provides rapid,reversible remote control of protein function that is selective withoutorthogonal chemistry.

Methods

Synthesis of iGluR6 Tethered Agonist MAG-2.

MAG-2 was synthesized using chemistry similar to that previouslydescribed for MAG-1 (See Example 2 for synthesis of MAG-1.).

All non-aqueous reactions were performed using flame- or oven-driedglassware under an atmosphere of dry nitrogen. Commercial reagents wereused as received. Non-aqueous reagents were transferred under nitrogenwith a syringe or cannula. Solutions were concentrated in vacuo on aBuchi rotary evaporator. Diisopropylethylamine (DIPEA) was distilledfrom calcium hydride prior to use. Tetrahydrofuran (THF) and methylenechloride (CH₂Cl₂) were passed through a column of activated aluminaunder N₂-pressure prior to use. N,N-Dimethyl formamide (DMF) wasdegassed with a stream of N₂, dried over molecular sieves, and usedwithout further purification. Chromatographic purification of productswas accomplished using flash column chromatography on ICN 60 32-64 meshsilica gel 63 (normal phase) or Waters Preparative C18 125 Å 55-105 μmsilica gel (reversed phase), as indicated. Thin layer chromatography(TLC) was performed on EM Reagents 0.25 mm silica gel 60-F₂₅₄ plates.Visualization of the developed chromatogram was performed usingfluorescence quenching, KMnO₄, ceric ammonium molybdate (CAM), or iodinestains. IR spectra were measured with a Genesis FT-IR spectrometer bythin film or Avatar 370 FT-IR by attenuated total reflectance accessory.Optical rotations were measured using a Perkin-Elmer 241 Polarimeter at25° C. and 589 nm. ¹H and ¹³C NMR spectra were recorded in deuteratedsolvents on Bruker AVB-400, AVQ-400, or DRX-500 spectrometers andcalibrated to the residual solvent peak. Multiplicities are abbreviatedas follows: s=singlet, d=doublet, t=triplet, q=quartet, m=multiplet,app=apparent, br=broad.

To a solution of azodianiline (2.00 g, 9.4 mmol),1-Hydrozy-benzotriazole hydrate (HOBt) (1.91 g, 14.1 mmol),Diisopropylamine (DIPEA) (6.54 mL, 37.6 mmol), andN-Ethyl-N′-(3-dimethyldiaminopropyl)-carhodiimide.HCl (EDCI) (2.34 g,12.2 mmol) in CH₂Cl₂ (260 mL) was added a solution of Boc-Gly-Gly-OH(2.39 g, 10.3 mmol) in CH₂Cl₂ (50 mL). The mixture was stirred at roomtemperature for 12 h. The mixture was diluted with CH₂Cl₂ (500 mL) andwashed with a saturated NaHCO₃ solution (2×500 mL) and brine (2×500 mL).The organic layer was dried over Na₂SO₄, filtered, and concentrated.Purification by normal phase chromatography (dry loaded, 10:0→20:1CH₂Cl₂:MeOH) gave 4 (878 mg, 58% based on recovered azodianiline) as anorange solid. Data for 4: R_(f) 0.28 (95:5 CH₂Cl₂: MeOH); mp 190-192°C.; UV λ_(max) (MeOH): 394 nm; IR: 3397, 3365, 3273, 1712, 1703, 1651,1598, 1613, 1550 cm⁻¹; ¹H NMR (DMSO-d₆, 400 MHz) δ 10.09 (s, 1H), 8.20(m, 1H), 7.72 (m, 4H), 7.61 (d, 2H, J=9 Hz), 7.08 (m, 1H), 6.65 (d, 2H,J=9 Hz), 6.02 (s, 2H), 3.92 (d, 2H, J=6 Hz), 3.60 (d, 2H, J=6 Hz), 1.38(s, 9H); ¹³C NMR (DMSO-d₆, 125 MHz) δ 173.3, 169.7, 158.8, 153.3, 150.7,145.7, 140.7, 126.0, 123.7, 121.4, 115.2, 81.0, 45.0, 44.0, 28.7; HRMS(FAB) Calc for C₂₁H₂₇N₆O₄ (M)⁺: 427.209379 Found: 427.210320.

To a solution of 4 (1.00 g, 1.53 mmol) in a 5:1 mixture of CH₂Cl₂:MeOH(90 mL) was added trifluoroacetic acid (90 mL). The mixture was stirredfor 4 h at room temperature, concentrated, and triturated with diethylether (2×100 mL) to yield 5 (1.01 g, 98%) as a purple solid. Data for 5:mp 174-175° C.; UV λ_(max) (MeOH): 394 nm; IR: 3269, 3080, 1666, 1599,1575, 1540, 1506 cm⁻¹; ¹H NMR (MeOH-d₄, 500 MHz) δ 7.83 (m, 4H), 7.75(d, 2H, J=9 Hz), 7.12 (d, 2H, J=7 Hz), 4.13 (s, 2H), 3.80 (s, 2H); ¹³CNMR (DMSO-d₆, 125 MHz) δ 169.4, 169.1, 149.7, 148.7, 141.8, 126.2,124.1, 121.2, 119.7, 43.9, 41.5; HRMS (FAB) Calc for C₁₆H₁₉N₆O₂ (MH)⁺:327.156949. Found: 327.156620.

To a solution of 5 (913 mg, 2.07 mmol), HOBt (421 mg, 3.11 mmol), DIPEA(1.44 mL, 8.29 mmol), and EDCI (517 mg, 2.69 mmol) in CH₂Cl₂ (100 mL)was added a solution of 7 (854 mg, 2.49 mmol) in CH₂Cl₂ (25 mL). Themixture was stirred at room temperature for 12 h. The mixture wasdiluted with CH₂Cl₂ (300 mL) and washed with a saturated NaHCO₃ solution(2×300 mL) and brine (2×300 mL). The organic layer was dried overNa₂SO₄, filtered, and concentrated. Purification by normal phasechromatography (95:5→90:10 CH₂Cl₂:MeOH) gave 6 (442 mg, 33%) as anorange solid. Data for 6: R_(f) 0.29 (95:5 CH₂Cl₂: MeOH); mp 124-126°C.; [α]_(D)=−54.0 (c 0.1, in MeOH); UV λ_(max) (MeOH): 394 nm; IR: 3356,2980, 2922, 2844, 1652, 1597, 1558 cm⁻¹; ¹H NMR (MeOH-d₄, 400 MHz) δ7.76 (s, 4H), 7.69 (d, 2H, J=9 Hz), 6.73 (d, 2H, J=9 Hz), 4.55 (d, 1H,J=10 Hz), 4.19 (m, 2H), 4.05 (m, 2H), 3.89 (m, 2H), 2.60 (m, 1H), 2.33(m, 2H), 2.19 (m, 1H), 2.00 (m, 1H), 1.85 (m, 1H), 1.70 (m, 2H), 1.44(s, 9H), 1.40 (m, 1H), 1.24 (t, 3H, J=7 Hz); ¹³C NMR (MeOH-d₄, 100 MHz)δ 178.0, 176.6, 173.1, 172.6, 169.8, 153.4, 150.8, 150.7, 145.7, 140.9,126.2, 124.0, 121.5, 115.3, 84.8, 63.0, 58.8, 44.3, 42.7, 36.4, 30.9,29.0, 28.2, 23.9, 14.7; HRMS (FAB) Calc for C₃₂H₄₀N₇O₈ (MH)⁺:651.301662. Found: 651.300110.

To a solution of Fmoc-Gly-OH (74.5 mg, 0.250 mmol) and oxalyl chloride(150 of 2.0 M solution in THF, 0.300 mmol) in CH₂Cl₂ (2.0 mL) was addedone drop of DMF. After stirring for 1 h at room temperature the mixturewas concentrated. The resulting acid chloride was redissolved in THF(3.0 mL) and added via cannula to a solution of 6 (81.7 mg, 0.125 mmol),DIPEA (109 μL, 0.625 mmol), and 4-(Dimethylamino)-pyridine (DMAP) (1.5mg, 0.013 mmol) in THF (7.0 mL). After stirring 10 min at 0° C., themixture was warmed to room temperature and stirred an additional 3 h.The mixture was then diluted with CH₂Cl₂ (100 mL) and washed with asaturated solution of NaHCO₃ (2×100 mL) and brine (2×100 mL). Theorganic layer was dried over Na₂SO₄, filtered, and concentrated.Purification by normal phase chromatography (90:10:0.6:0.6CH₂Cl₂:MeOH:AcOH:H₂O) gave the Fmoc-glycine adduct as an orange solidthat was sufficiently pure for further reaction. To a solution of thiscompound in DMF (8.0 mL) was added piperidine (100 μL, 1.01 mmol). Afterstirring for 6 h at room temperature, the mixture was concentrated andpurified by normal phase chromatography (90:10:0.6:0.6→80:20:3:3CH₂Cl₂:MeOH:AcOH:H₂O) to yield 8 (56.8 mg, 64%) as an orange solid. Datafor 8: mp>200° C. dec; [α]_(D)=−24.0 (c 0.1, in DMSO); UV λ_(max)(MeOH): 378 nm; IR: 3281, 2981, 1780, 1741, 1595, 1541 cm⁻¹; ¹H NMR(MeOH-d₄, 500 MHz) δ 7.85 (m, 6H), 7.77 (d, 2H, J=9 Hz), 4.56 (d, 1H,J=9 Hz), 4.20 (m, 2H), 4.06 (s, 2H), 3.90 (s, 2H), 3.84 (s, 2H), 2.60(m, 1H), 2.33 (m, 2H), 2.20 (m, 1H), 2.02 (m, 1H), 1.76 (m, 1H), 1.71(m, 2H), 1.44 (s, 9H), 1.43 (m, 1H), 1.25 (t, 3H, J=7 Hz); ¹³C NMR(DMSO-d₆, 100 MHz) δ 174.7, 172.7, 172.5, 171.4, 169.7, 168.2, 148.8,147.7, 147.6, 141.6, 141.5, 123.4, 123.3, 119.4, 119.2, 82.3, 61.2,56.7, 45.6, 42.9, 42.3, 40.8, 34.9, 29.5, 27.5, 22.5, 22.3, 14.0; HRMS(FAB) Calc for C₃₄H₄₅N₈O₉ (M)⁺: 709.330951. Found: 709.332180.

To a solution of 8 (152 mg, 0.214 mmol) in THF (8.0 mL) at 0° C. wasadded 1.0 M LiOH (8.0 mL). After stirring for 1 h, the mixture wasacidified to pH 2 with 1 M HCl, THF was removed in vacuo, and themixture was purified by reversed phase chromatography (5:0→4:1 0.1%formic acid in H₂O:MeCN) to yield 9 (95.1 mg, 63%) as a yellow solid.Data for 9: mp>230° C. dec; [α]_(D)=−20.0 (c 0.1, in DMSO); UV λ_(max)(DMSO): 378 nm; IR: 3285, 3070, 2933, 1666, 1598, 1538, 1501 cm⁻¹; ¹HNMR (DMSO-d₆, 400 MHz) δ 10.21 (s, 1H), 8.37 (m, 1H), 8.22 (m, 2H), 7.84(m, 8H), 7.55 (m, 1H), 6.88 (m, 1H), 6.81 (m, 1H), 3.92 (m, 2H), 3.76(m, 1H), 3.72 (m, 2H), 3.55 (s, 2H), 2.35 (m, 1H), 2.14 (m, 2H), 1.91(m, 1H), 1.58 (m, 1H), 1.48 (m, 4H), 1.35 (s, 9H); ¹³C NMR (DMSO-d₆, 100MHz) δ 176.4, 174.2, 172.8, 169.7, 169.3, 168.2, 155.5, 147.8, 147.7,141.6, 141.2, 123.5, 123.4, 119.4, 77.9, 52.5, 43.6, 42.9, 42.3, 41.6,35.0, 33.3, 32.0, 28.2, 22.8; LRMS (ESI)⁻ Calc for C₃₂H₄₁N₈O₁₀ (M-H)⁻:697.3. Found: 697.2.

To a solution of 9 (36 mg, 0.052 mmol) in a saturated solution of NaHCO₃(2.5 mL) was added finely ground N-methoxycarbonylmaleimide (36 mg,0.232 mmol) under vigorous stirring. After 30 min at 0° C., the mixturewas diluted with THF (2.5 mL) and warmed to room temperature. After 1 h,the mixture was acidified to pH 1-2 with an aqueous solution of 1.0 MH₂SO₄ and extracted with EtOAc (2×25 mL). The combined organic layerswere dried over Na₂SO₄, filtered, and concentrated. Purification bynormal phase chromatography (86:14:1.5:1.5 CH₂Cl₂:MeOH:AcOH:H₂O) gave 10(17.1 mg, 43%) as a yellow solid. Data for 10: R_(f) 0.27 (86:14:1.5:1.5CH₂Cl₂:MeOH:AcOH:H₂O); mp>230° C. dec; [α]_(D)=−17.0 (c 0.1, in MeOH);UV λ_(max) (MeOH): 377 nm; IR: 3298, 3088, 2931, 1705, 1683, 1538 cm⁻¹;¹H NMR (DMSO-d₆, 400 MHz) δ 10.66 (s, 1H), 10.21 (s, 1H), 8.36 (t, 1H,J=6 Hz), 8.23 (t, 1H, J=6 Hz), 7.84 (m, 6H), 7.75 (d, 2H, J=9 Hz), 7.15(s, 2H), 4.32 (s, 2H), 3.92 (d, 2H, J=4 Hz), 3.78 (m, 1H), 3.73 (d, 2H,J=5 Hz), 2.37 (m, 1H), 2.14 (m, 2H), 1.92 (m, 1H), 1.48 (m, 5H), 1.35(s, 9H); ¹³C NMR 176.4, 174.3, 172.7, 170.7, 169.6, 168.2, 165.3, 155.5,147.9, 147.7, 141.5, 141.0, 135.0, 123.5, 119.4, 119.3, 77.9, 52.5,42.9, 42.2, 41.6, 40.5, 35.0, 33.3, 32.0, 28.2, 22.8; LRMS (ESI)⁻ Calcfor C₃₆H₄₁N₈O₁₂ (M-H)⁻: 777.3. Found: 777.2.

To a flask containing solid 10 (29 mg, 0.038 mmol) was added a saturatedHCl solution in EtOAc (17.0 mL). After stirring at room temperature for2 h, the resulting purple solid was triturated with ethyl ether (2×60mL) to yield 2 (19.7 mg, 73%). Data for 2: mp>230° C. dec; [α]_(D)=−12.0(c 0.1, in DMSO); UV λ_(max) (10% DMSO in H₂O): 364 nm; IR: 3298, 3087,2930, 1706, 1683, 1652, 1539 cm⁻¹; ¹H NMR (DMSO-d₆, 500 MHz) δ 10.70 (s,1H), 10.25 (s, 1H), 8.28 (m, 4H), 8.21 (m, 1H), 7.84 (m, 6H), 7.76 (d,2H, J=9 Hz), 7.16 (s, 2H), 4.32 (s, 2H), 3.93 (d, 2H, J=6 Hz), 3.79 (m,1H), 3.74 (d, 2H, J=6 Hz), 2.57 (m, 1H), 2.17 (m, 2H), 2.12 (m, 1H),1.75 (m, 1H), 1.52 (m, 4H); ¹³C NMR (DMSO-d₆, 125 MHz) δ 175.6, 172.5,170.9, 170.7, 169.9, 168.2, 165.4, 147.8, 147.7, 141.6, 141.1, 135.0,123.5, 119.4, 119.3, 64.9, 50.8, 42.8, 42.1, 34.9, 31.7, 31.2, 22.3,15.2; HRMS (ESI)⁺ Calc for C₃₁H₃₄N₈O₁₀ (M)⁺: 678.23979. Found:678.23954.

To a solution of 2 (2.3 mg, 3.5 mmol) dissolved in a saturated solutionof NaHCO₃ (200 mL) at room temperature was added 13ME (20 μL). Afterstirring for 1 h, the mixture was concentrated and was purified byreversed phase chromatography (10:0→7:3 H₂O:MeCN) to yield 11 as ayellow solid. Data for 11: LRMS (ESI)⁻ Calc for C₃₁H₃₆N₇O₁₀S (M-H)⁻:698.2. Found: 698.1.

Photostationary State Determination by NMR

Cis-trans photoisomerization of MAG-1 conjugated with β-mercaptoethanol(see Supplementary Methods online) was studied by ¹H NMR, with in situirradiation of sample using a Polychrom V system monochromator (TillPhotonics) containing a 150 W Xenon short arc lamp with an output rangeof 320-680 nm (29). The half-power bandwidth was 14 nm. A 500 μl aliquotof a 100 μM sample in D₂O was prepared in a screw-capped 528-TR-7 NMRtube (Wilmad). A FT-600-UMT fiber optic cable (NA 0.39) (Thorlabs) wascoupled at one end to the monochromator using a custom fitting and theother end inserted into the NMR tube a few millimeters above thesolution. Each sample was irradiated at the desired wavelength for 30minutes to reach the photostationary state and 480 scans were requiredto obtain reasonable signal-to-noise ratios. The output from the fiberoptic cable between 340-480 nm ranged from 0.3-9.0 μW/cm². Data wereprocessed by isolating aromatic cis-trans ¹H signals and summing theirintegrals to 1.0. All data were obtained in triplicate and averaged.This analysis will be detailed further in a future paper by Banghart, M.R. et al. (in preparation).

Introduction of Cysteine Residues in the Glutamate Binding Domain ofiGluR6.

Cysteine point mutations were introduced to the iGluR6 DNA, containing Qat the position 621 RNA editing site (30) using the QuickChangesite-directed mutagenesis kit (Stratagene). The following PCR profilewas used: one cycle (95° C. for 30 s); 20 cycles (95° C. for 30 s, 55°C. for 1 min, 68° C. for 12 min). The forward and reverseoligonucleotide sequences designed for the L439C mutant were

(SEQ ID NO: 1) 5′-GATTGTTACCACCATTTGCGAAGAACCGTATGTTCTG-3′; and(SEQ ID NO: 2) 5′-CAGAACATACGGTTCTTCGCAAAATGGTGGTAACAATC-3′.Cell Culture and Transfection

HEK293 cells were plated at approximately 3×10⁶ cells/ml onpoly-L-lysine-coated glass coverslips (Deutsche Spiegelglas, CarolinaBiological) and maintained in DMEM with 5% fetal bovine serum, 0.2 mg/mlstreptomycin, and 200 U/ml penicillin at 37° C. Cells were transientlytransfected with various plasmids using lipofectamine 2000 (Invitrogen).The amount of total transfected iGluR6 DNA and EYFP fusion DNA per 2 mlwell was fixed at 4 μg and 200 ng, respectively. All recordings werecarried out 36 to 48 h after transfection.

Conjugation of MAG Compounds.

To conjugate MAG-1 and MAG-2 to iGluR6-L439C, the compounds were dilutedin the HEK cell control solution to 100 nM-200 μM from concentratedstock solutions in DMSO (final DMSO concentration being 0.1% at most).These solutions were irradiated for 1 hour with 365 nm light using ahandheld UV lamp (UVP model UVGL-25 (multiband 254 nm/365 nm), UplandCA).

Calcium Imaging

Cells were washed in PBS and loaded with 5 μM Fura-2-AM (MolecularProbes) for 30 min. Changes of [Ca²⁺], in individual cells were measuredas intracellular Fura2 fluorescence intensity using mercury arc lampillumination and alternating excitation with band pass filters of 350 nmand 380 nm during 66 ms at 5-20 s intervals and detecting emission at510 nm (31). Fluorescence was monitored on an inverted microscope system(Nikon). Images were captured with a CCD camera using the ImagingWorkbench software, which was also used to irradiate the cells at 380and 500 nm during 1-2 min in order to produce photoisomerization of MAG.Measurements were performed in a control solution containing (in mM):135 NaCl, 5.4 KCl, 0.9 MgCl₂, 1.8 CaCl₂, 10 glucose and 10 HEPES at pH7.6. Cells were preincubated for 10 min in control solution containing300 mg/l Concanavalin A type IV (Sigma) in order to blockdesensitization (32, 33). L-Glutamate was applied as reported in textand figures. The results are representative data from multiple cells inat least two independent cultures.

Whole-Cell Patch Clamping

Patch clamp recordings were carried out using an Axopatch 200A amplifierin the whole cell mode. Cell voltage was held at −60 mV. Pipettes hadresistances 4-8 MΩ and were filled with a solution containing (in mM):145 CsCl, 5 EGTA, 0.5 CaCl₂, 1.0 MgCl₂ and 10 HEPES at pH7.2. Theextracellular solution and concanavalin preincubation were as in calciumimaging experiments. Blocking experiments were carried out with DNQXdisodium salt (Tocris) diluting a 4 mM stock solution in theextracellular solution, up to 100 nM. Leak was subtracted under 500 nm,4 mM DNQX. For each DNQX concentration, relative response to 380 nm wascalculated as I (380 nm, DNQX)/I (380 nm, control), and relativeresponse to 500 nm was calculated as I (500 nm, DNQX)/I (380 nm,control). Glutamate was added as indicated in the figures. Illuminationwas applied using a TILL Photonics Polychrome II monochromator through a60×/1.2 W objective (power output: 12.4 W/m² irradiance; 500 nm asmeasured with a Newport optical power meter). Data was recorded withpClamp software, which was also used to automatically control themonochromator by means of sequencing keys.

Results

Modular Photoswitchable Tethered Ligands

The photoswitchable tethered ligand was designed to possess a maleimidefor conjugation to a cysteine residue on the exterior of the LBD, aglutamate analog, and an azobenzene linker in between enablingreversible state-dependent control over the reach of the glutamateanalog (Example 2). The glutamate analog was chosen based on previouslyestablished structure-activity relationships of the selective iGluRagonists (2S,4R)-4-allyl-glutamate (LY310214) and(2S,4R)-4-methyl-glutamate (SYM 2081) (15, 16, 17), and on our noveliGluR6 agonist, termed the “tether model” (3; FIG. 10 b) (Example 2).The modularity of the design allows for the introduction of additionalglycine units in the tether with minimal synthetic investment. Initialstudies were based upon models of docking MAG-1 in the iGluR6-MeGlucrystal structure (16), while the exact tether length required foroptimal activation remained unknown. Following the synthesis of MAG-1,the elongated MAG-2 was synthesized using chemistry analogous to thatdescribed in Example 2. Different length MAGs allow for the study oftether length dependence on channel activation and agonist binding usingreadily accessible and minimally disruptive amino acid building blocks.The success of the MAG design, and the ease with which it can bemodified, opens the possibility of replacement of the glutamate moietyfor other iGluR agonists or antagonists, or application to othersimilarly functioning allosteric proteins with well defined ligandbinding modes.

FIGS. 10A and 10B. Modular photoswitchable tethered ligands.

(A) The light-gated glutamate receptor operates by reversibly binding ofthe photoswitchable agonist MAG (Example 2) which is attached covalentlyto a cysteine introduced in the ligand binding domain of the receptor.The ribbon structure of apo-iGluR2 (Protein Data Bank accession code 1FTO) (18) is shown on the left, together with the ball-and-stickstructure of MAG in the extended (trans) and unbound conformation. Under380 nm illumination MAG-1 can activate the receptor as is shown on theright with cis-MAG-1 docked on the structure of iGluR6 in complex with(2S,4R)-4-methyl glutamate (Protein Data Bank accession code 1FTO) (19).Photoswitching is reversible with 500 nm illumination. (B) The structureof MAG-1 (Example 2) can be elongated by introducing an additionalglycine unit (MAG-2). Compound 3 is a non-photoswitchable MAG-1 analogand an iGluR6 agonist termed the “tether model”.

Photostationary State Determination by NMR

In the thermally relaxed state, azobenzene exists almost entirely in thetrans configuration (20). Upon illumination, a mixture is generated,with a fraction of the azobenzene in the cis configuration and the restin trans. The balance between cis and trans (the photostationary state)depends on the wavelength. The cis population is maximally populated inthe near UV, and trans population is maximally populated in the visiblerange of the light spectrum. Usually absorbance is used to determine thefraction of azobenzene in the two states. This requires determination ofthe UV and visible spectra of the two isomers, which partially overlap.Here we used a novel approach of NMR spectroscopy to distinguish betweenthe two isomers. NMR was used to determine the ratio of cis- to transMAG-1 conjugated to β-mercaptoethanol between 340 and 500 nm, at 20 nmincrements (FIG. 11 a). Optimal wavelengths for cis and transpopulations were found to be 380 and 500 nm, respectively. At 380 nm93.0±0.6% of MAG-1 is in the cis-state and at 500 nm 83.0±0.6% of MAG-1is in the trans-state.

Spectral Sensitivity of Photoresponse Produces an Analog Output

In order to quantify the relationship between the photostationary stateof MAG-1 in solution and after conjugation to iGluR6-L439C, the currentamplitude and switching kinetics were measured as a function ofwavelength. Activation was examined by stepping wavelengths from maximalsteady-state deactivation (500 nm) to a series of shorter wavelengths.The step duration was selected to be 10 s, long enough for currents toreach steady state. Deactivation was examined by starting at thewavelength of maximal steady-state activation (380 nm) and stepping tolonger wavelengths (FIG. 11 b). The activation and deactivationcomponents were each well fit with a single exponential (FIG. 11 c). Theactivation spectrum is centered at 380 nm (FIG. 11 d “On”), and fallsoff steeply at higher and lower wavelengths. The deactivation spectrumis broader and is centered at −500 nm (FIG. 11 d “Off”), withwavelengths between 460 and 560 nm yielding maximal deactivation. TheNMR-based determination of the photostationary states of MAG-1 insolution, over the wavelength range of 320-500 nm, closely match theaction spectrum of channel activation when MAG-1 is conjugated to thechannel protein (FIG. 11 a). Furthermore, the on and off rates werefastest at wavelengths between 380 nm and 500 nm (FIG. 11 e).

FIGS. 11A-E. Photostationary State Determination by NMR and SpectralSensitivity of Photoresponses.

(A) Fraction of MAG-1 in the cis form determined from NMR spectroscopy.Maximal wavelengths for cis and trans populations are 380 and 500 nmrespectively. (B) Wavelength dependence of photoresponses ofiGluR6-L439C conjugated to MAG-1, measured by whole-cell patch clamp.The current vs. time traces and corresponding wavelength step protocolused to record action spectra are indicated. The first set of steps(activation spectrum) start at the wavelength of maximal deactivation(500 nm) and span UV illuminations of increasing wavelength. The secondset of steps (deactivation spectrum) start at the wavelength of maximalactivation (380 nm) and span visible illuminations of increasingwavelength. (C) Each temporal trace can be fitted with a singleexponential function whose amplitude and time constant is used to buildthe action spectra. (D) The activation spectrum (“ON”) is centered on380 nm and falls off rather steeply at higher and lower wavelengths. Thedeactivation spectrum (“OFF”) is wider, with maximal amplitude between460 nm and 560 nm. (E) Wavelength dependence of photoswitch rate. Timeconstants τ_(ON) and τ_(OFF) from fits of traces in (b) are representedas switch rates (1/T).

Thermal Relaxation of MAG

Experimentally, it may be advantageous to control channel openingwithout continuous irradiation. In such situations, a single activatingpulse of UV light would be used to initiate activation for extendedperiods of time (minutes). As such, the MAGs were designed with a4,4′-azodianiline scaffold modified with amide linkages to the glutamateand maleimide moeities of the molecule. These amide-based azobenzenecores are known to possess half-lives of minutes for the rate of thermalrelaxation from the cis-state to the lower energy trans-state in thedark (21).

The rate of thermal relaxation in the dark of free MAG-1 from cis totrans was measure; a half-life of 17.65±0.03 min was obtained (FIG. 12a). The spontaneous deactivation in the dark of iGluR6-L439C channelsthat were conjugated with MAG-1 was examined. Following activation witha 5 s pulse of illumination at 380 nm, there was a 25% decrease inchannel current after ten minutes in the dark (FIG. 12 b), agreeingclosely with the observed half-life of MAG-1 in solution. Similarobservations were made on iGluR6-L439C channels that were conjugatedwith MAG-2. The significance of persistent channel activity in the darkafter a brief pulse of illumination is that long-lasting currents can bemaintained in absence of irradiation, thus reducing photobleaching ofthe azobenzene, photo-damage to the protein and photo-toxicity to cells.

FIGS. 12A and 12B. Thermal relaxation of MAG.

(A) Rate of thermal relaxation in the dark of free MAG-1 from cis totrans, measured by absorbance at 360 nm. Traces are exponential anddisplay a half-life of 17.65±0.03 min. (B) Spontaneous rate ofdeactivation of iGluR6-L439C conjugated to MAG-1, after activation witha 5 s pulse at 380 nm. The current decreases 25% after 10 min.

MAG Conjugation to iGluR6-L439C Occurs by Affinity Labeling

In our first study (14), a model of MAG-1 in the cis-state was dockedonto the crystal structure of iGluR6 in complex with(2S,4R)-4-methyl-glutamate. When the glutamate moiety was fit in theagonist binding site, the maleimide end of MAG-1 was able to reach aminoacid 439, where the introduced cysteine permits conjugation and yields alight-gated channel (Example 2) (FIG. 10 a). This provided a vividpicture of the photoactivated state, and raised the question of whetheroccupancy of the binding site by MAG-1 would enhance the conjugationefficiency of the maleimide to the cysteine at position 439 by affinitylabeling. Affinity labeling has been observed in a variety of systems(22), including in the conjugation of tethered blockers to the Shaker K⁺channel (23), which served as a basis for the development of thephotoswitchable SPARK channel (13).

In order to investigate the nature of MAG conjugation, affinity labelingwas interfered with in two ways. In a first experiment, it was askedwhether labeling could be hindered by using visible light to favor thetrans-state of MAG-1 conformation, which is expected to extend themaleimide away from cysteine 439 when bound (FIG. 13 a). The efficiencyof MAG-1 conjugation from the amplitude of photo-responses was evaluatedusing calcium imaging to detect the activation of the calcium permeantiGluR6 channels, as described in Example 2. Incubation with 100 nM MAG-1under 380 nm light (favoring the cis-state) produced larger subsequentphoto-responses than did incubation under 500 nm light (favoring thetrans-state) (FIG. 13 c). This is consistent with state-dependentconjugation, which is expected to better position the maleimide near theengineered cysteine when MAG-1 is bound and is in the cis-state.

In a second experiment, affinity labeling was interfered with byoccupying the ligand binding site with saturating free glutamate duringthe incubation period (FIG. 13 b). Incubation was carried under 380 nmlight to favor the cis-state, as shown above. Incubation of iGluR6-L439Cwith 100 nM MAG-1 in the absence of free glutamate for 15 min producedsignificantly larger subsequent photo-responses than did incubation inthe presence of 300 μM glutamate (FIG. 13 d). The disruption of affinitylabeling in the presence of glutamate is consistent with competitionbetween the glutamate end of MAG and free glutamate for the ligandbinding site.

Together these experiments demonstrate that at low concentrations MAGconjugation operates by affinity labeling. The ability to controlphotoswitch attachment under UV light opens the possibility of selectivelabeling only in illuminated regions of a sample.

FIGS. 13A-D. MAG-1 Conjugation to iGluR6-L439C Occurs by AffinityLabeling.

MAG-1 conjugation at 100 nM can be interfered by two means: (A) favoringthe trans-configuration with 500 nm illumination, which orients themaleimide away from cysteine 439 when the glutamate is bound at thebinding site, and (B) occupying the binding site with free glutamate,thus preventing docking of MAG-1. (C) Photoresponses obtained by calciumimaging after MAG-1 conjugation under the conditions shown in (A). Weakresponses are obtained after MAG-1 conjugation at 100 nM under visibleillumination (MAG-1 in trans, maleimide away from cysteine 439), but asubstantial increase in photoresponses is observed after conjugationunder UV (MAG in cis; maleimide near cysteine 439). (D) Weak responsesare obtained after MAG-1 conjugation at 100 nM in the presence of 300 μMglutamate (ligand binding pocket occupied), but are substantiallyincreased after MAG-1 conjugation at 100 nM in the absence of glutamate.

Concentration Dependence of MAG Conjugation

Previous work had demonstrated that the photo-currents of iGluR6-L439Cconjugated to MAG-1 are smaller than the currents induced by saturatingglutamate (300 μM). It was asked whether the partial activation byiGluR6-L439C-MAG-1 is due to incomplete MAG conjugation. Usingwhole-cell patch clamping labeled channels were tested with MAG-1 for 1hour under 380 nm illumination, using concentrations of 0.1, 10, and 200μM, with the final concentration being the solubility limit of MAG-1.The average photo-current increased with increasing concentration.Relative to the currents evoked by saturating glutamate, the opticalactivation of iGluR6-L439C-MAG-1 at 380 nm produced currents of 21±8%,54±20%, and 71±19% at 0.1, 10, and 200 respectively.

It was considered what these values would mean for the activation of atetrameric protein, with four ligand-binding sites. With completeconjugation, and under optimal excitation at 380 nm illumination tomaximize the activating state, each molecule of MAG will spend ˜93% ofits time in the cis-state. This means that in the tetrameric channel,four LBDs will be activated at the same time 75% (0.93) of the time.Although liganding of only a fraction of a channel's LBDs stillgenerates some current in iGluR5 (24, 25) our results suggest that atthe higher concentration we are close to full labeling and that MAG-1conjugated to iGluR6-L439C functions as an agonist that is similar inpotency to glutamate.

MAG Functions as a Full Agonist

It was asked whether, when attached to the channel, MAG-1 operates as afull agonist. The “tether model” was developed (3; FIG. 10 b) on whichMAG is based to be a full agonist, i.e. to be as effective at activatingchannels as glutamate itself (Volgraf et al., 2006). When partialagonists bind they allow only partial closure of the LBD and thus onlypartial channel activation (26). It was asked whether the MAG linkerbetween the maleimide and the glutamate would be constrained in such away when the maleimide is conjugated to the introduced cysteine that itwould partially obstruct closure of the LBD. In the presence of 300 μMglutamate, photoswitching to cis-MAG-1 will compete with glutamate forthe binding site and replace some of the free glutamate with MAG-1. IfMAG-1 were a partial agonist this would reduce the current (i.e. act asa partial antagonist).

This prediction was tested and found that iGluR6-L439C conjugated with100 μM MAG-1 for 15 minutes (i.e. expected to yield substantial, butlikely incomplete conjugation, see above) did not show a sign of partialantagonism. Rather than decrease currents, photo-activation(isomerization to cis at 380 nm) of iGluR6-L439C-MAG-1 in the presenceof glutamate slightly increased the current (FIG. 14). This observationargues that MAG-1 is actually a slightly better agonist than glutamate.

FIG. 14. MAG functions as a full agonist. Patch clamp trace showingresponses to 380 nm illumination that are lower than glutamate 300 μMresponses. When photoresponses are elicited during glutamate perfusion,they result in a slight current increase rather than a decrease,indicating that when MAG displaces glutamate it acts as a full and notpartial agonist.

High Effective Local Concentration of MAGs

It was shown in Example 2 that agonist 3, a MAG analogue lacking amaleimide and full-length azobenzene, has an EC₅₀ of 180 μM foractivating iGluR6. Although compound 3 possesses a relatively weakaffinity, the local concentration of the glutamate end of cis-MAG-1 whenconjugated to iGluR6-L439C is expected to be very high based on itsshort tether.

To test this idea, the effective concentration of the glutamate end ofMAG-1 was estimated using the competitive antagonist DNQX (27). DNQXinhibits iGluR activation by occupying the glutamate binding site andstabilizing an open conformation of the LBD (FIG. 15 a) (28). Theability of DNQX to competitively inhibit the responses of iGluR6-L439Cto light-activation with MAG-1, and to perfusion with compound 3 orglutamate, was examined. DNQX inhibited the response to MAG-1 at 380 nmillumination in a concentration dependent manner and was completelyreversible upon washout (FIG. 15 b). The inhibition curve had a 50%inhibition (IC₅₀) of the cis-state light response at 220±65 μM DNQX(FIG. 15 c). Even at the DNQX solubility limit of 4 mM the block of thephoto-current was incomplete.

To calculate the local concentration of the glutamate end of MAG-1 DNQXcompetition versus compound 3, the closest soluble MAG analogue, wasexamined. The concentration dependence of DNQX inhibition was measuredusing two known concentrations of the tether model (3 mM and 10 mM, thelatter being the solubility limit) in order to extrapolate effective MAGconcentrations from their DNQX IC₅₀. At 3 mM and 10 mM concentrations ofcompound 3 DNQX IC₅₀ values of 39±15 μM and 202±26 μM, respectively,were obtained (FIG. 15 c). Thus, inhibition by DNQX reveals that in thecis-state the glutamate moiety of MAG-1 has an effective concentrationof 12.5 mM (FIG. 15 d). Such a high effective concentration (50-foldgreater than the EC₅₀ of compound 3) suggests that the photo-switchedtethered ligand functions as designed on the channel, generating a veryhigh effective local concentration in the cis-state.

The antagonist competition experiment revealed the existence of a basalcurrent of ˜20% at 500 nm that was blocked by DNQX. The IC₅₀ value ofblock of this basal current by DNQX was quantified, and found to be 7±2μM (FIG. 15 c). This value indicates an effective glutamateconcentration of 0.5 mM, that is ˜30-fold lower than what we measured at380 nm (FIG. 12 d), supporting the model that light turns the channel onand off because photoisomerization of the azobenzene repositions MAG-1and changes its ability to bind in the glutamate binding pocket.

FIGS. 15A-D. Effective Local Concentration of MAG-1 is ˜12 mM.

(A) The competitive antagonist DNQX inhibits iGluR activation byoccupying the glutamate binding site without allowing clamshell closure.(B) Patch clamp current traces of iGluR6-L439C conjugated to MAG-1 showresponses to perfusion of glutamate 300 μM and to illumination. Thecorresponding wavelength-time traces are shown below. Perfusion of DNQXpartially inhibits photoresponses to 380 nm illumination and reveals abasal activation under 500 nm illumination Inhibition by DNQX isreversible upon washout after each DNQX perfusion. (C) Quantification ofDNQX inhibition of photoresponses and comparison to its effect oncompound 3. Current under 380 nm light (●) is inhibited by DNQX to 36%of total photoresponse (IC₅₀=220 μM DNQX) and the current under 500 nm(◯) is completely blocked, which reveals a basal activation ˜20% oftotal photoresponse, IC₅₀=7 μM DNQX. For comparison, DNQX blocksresponses to 10 mM tether model 3 (▪, IC₅₀=202 μM DNQX) and 3 mM tethermodel 3 (□, IC₅₀=39 μM DNQX). (D) Determination of the effectiveconcentration of MAG-1 as a function of the DNQX IC₅₀ values. The IC₅₀values for DNQX/tether model 3 are used to calibrate the localconcentration axis assuming a linear relationship (straight line), andyield 12.5 mM and 0.5 mM for MAG-1 under UV and visible respectively.

Tether Length Dependence on Channel Activation

The dependence of light-gating on tether length was investigated usingan elongated tethered ligand, MAG-2 (FIG. 16 a). It was found that foriGluR6-L439C conjugated to MAG-2 at 10 μM for 1 h the amplitude of thephotoresponse at 380 nm was 43±12% that of the current evoked bysaturating glutamate, slightly lower than what was measured foriGluR6-L439C-MAG-1 (FIG. 16 b). Competition studies oniGluR6-L439C-MAG-2 using DNQX yielded an IC₅₀ under 380 illumination of80±20 μM, indicating an effective concentration that was ˜3-fold lowerthan that observed for MAG-1. Consistent with the lower effectiveconcentration of MAG-2 in the cis-state, the basal activation was lowerthan for MAG-1 (FIG. 6 c) and high concentrations of DNQX completelyblocked the photo-current at 380 nm for iGluR6-L439C-MAG-2, even thoughblock was only partial for MAG-1 (FIG. 16 d).

FIGS. 16A-D. Tether Length Dependence on Channel Activation.

(A) DNQX titrations of iGluR6-L439C conjugated to MAG-1 (UV ●, vis ◯)and MAG-2 (UV ▴, vis Δ). (B) Amplitude of MAG-1 and MAG-2 photoresponsesafter conjugation at 10 μM for 1 h, compared to 300 μM glutamateresponses. (C) Basal activation of iGluR6-L439C conjugated to MAG-1 andMAG-2. (D) Residual photoresponse for MAG-1 and MAG-2 after 4 mM DNQX.

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Example 4 Photoswitchable Affinity Labels (PAL) Control Neuronal Firingof Endogenous Ion Channels

Methods

Cell Culture and Transfection.

HEK293 cells were grown in DMEM containing 5% fetal bovine serum, at 37°C., 7% CO₂. For electrophysiology, cells were plated at 12×10⁵ cells/cm²on poly-L-lysine coated coverslips and transfected using the calciumphosphate method. Recordings were performed 24-36 hours aftertransfection.

Hippocampal neurons were prepared from neonatal Sprague-Dawley ratsaccording to standard procedures (Goslin) and grown on poly-L-lysinecoated coverslips in MEM supplemented with 5% fetal bovine serum, B27(Invitrogen), glutamine and serum extender (BD Biosciences). Animal careand experimental protocols were approved by the UC Berkeley Animal Careand Use Committee. Recordings were performed 9-16 days after plating.The Shaker construct also contains an N-terminal deletion (Δ6-46) tominimize fast inactivation.

Brain Slice Preparation.

Parasagittal cerebellar slices were prepared using standard techniquesapproved by the UCLA Animal Care Committee. The cerebellum was removedfrom the cranium of a 14-20 day old Sprague-Dawley rat, mounted on anagar support and sectioned parasagitally using a vibrotome (LeicaVT-1000) while submerged in cold (<4° C.) artificial cerebrospinal fluid(aCSF) containing in mM: 119 NaCl, 26 NaHCO₃, 11 glucose, 2.5 KCl, 2.5CaCl₂, 1.3 MgCl₂, and 1 NaH₂PO₄ and saturated with 95% O₂ and 5% CO₂Following sectioning, the 300 μM slices were stored in 35° C. aCSF for30 minutes and brought to room temperature for subsequentelectrophysiological experiments.

PAL Attachment.

HEK293 and hippocampal cells were rinsed in extracellular solutioncontaining in mM: 138 NaCl, 1.5 KCl, 1.2 MgCl₂, 2.5 CaCl₂, 5 HEPES, 10glucose and incubated at 37° C. in the dark with 200-250 μM PAL for 15minutes. For slices, AAQ was diluted to 200 μM in aCSF and treatment wasperformed in the dark at room temperature for 10 minutes.

Live/Dead Assay.

After PAL treatment, hippocampal neurons were processed for the livedead assay (Molecular Probes) according to the manufacturer'sinstructions. Cells were counted in 4-8 fields for each treatmentcondition.

Electrophysiological Recording.

Recordings from HEK293 and hippocampal neurons were made in thewhole-cell patch clamp configuration using a PC-501A amplifier (WarnerInstruments). Pulse protocols and measurements were carried out withpCLAMP and a Digidata 1322 interface (Axon Instruments). Forvoltage-clamp experiment, cells were held at −70 mV. Data was recordedat 20 kHz and filtered at 2 kHz. Recording pipettes with 2.5-4 MΩresistance were filled with intracellular solution containing in mM: 10NaCl, 135 K-gluconate, 10 HEPES, 2 MgCl₂, 2 MgATP, 1 EGTA, pH 7.4.

For cerebellar slices, neuronal cell bodies were visualized using anupright microscope with a 40× water immersion lens and equipped with aninfrared-DIC enhancement. Loose-patch extracellular recordings wereperformed using an Axopatch 200B amplifier (Axon Instruments).Electrophysiological recordings were filtered at 1 KHz and digitized at2-4 KHz. Pipettes were typically 1.4-2.0 MΩ for recordings from PNs and3.0-3.5 MΩ for interneurons and filled with aCSF. For PN recordings,6,7-dinitroquinoxaline-2,3-[1H,4H]-dione (DNQX) and gabazine were addedwhen indicated at 20 μM and 10 μM respectively. Recordings from B/Scells were made in the presence of DNQX, gabazine and 5 μM[RS]-3-[2-carboxypiperazin-4-yl]-propyl-1-phosphonic acid (CPP). Allrecordings were performed at room temperature.

Illumination was provided using a xenon lamp (Sutter Instruments) withnarrow band pass filters (380 BP10 for UV and 500 BP5 for visible lightrespectively). Excitation filters were changed using a lambda 10-2filter changer (Sutter Instruments) controlled via pCLAMP software.

Percent photoswitching was defined as the difference between current inUV and visible light divided by current in UV. All data shown in bargraphs are averages±standard error of the mean.

Results

The PAL Approach.

The photoswitch used in SPARK channels is a derivative of thephotoisomerizable molecule azobenzene (AZO) (FIG. 17 a). Connected tothe AZO on one end is a cysteine-reactive maleimide (MAL) group, whichallows attachment to a specific cysteine that was introduced into anextracellular site on the Shaker protein, and on the other end aquaternary ammonium (QA) group, which can block the pore of K⁺ channels.The photoswitch molecule (MAL-AZO-QA, or for simplicity “MAQ”) isdesigned so that the QA can reach the pore and block ion conduction whenthe AZO is in its elongated trans form, but not in its bent cis form(FIG. 17 b). Exposure to 360-400 nm light photoisomerizes the AZO fromtrans to cis, unblocking the channel, whereas long wavelength light(450-520 nm) restores the blocked state by accelerating the reverse cisto trans conversion.

The PAL molecules that were designed for native K⁺ channels are similarto MAQ, with one important difference. Instead of maleimide, which iscommonly used as a cysteine-modifying reagent, PALs contain anelectrophilic group that was presumed would be more “promiscuous” (FIG.17 c), potentially reacting with a variety of nucleophilic amino acids.The following sequence of events when a PAL encounters a QA-sensitive K⁺channel (FIG. 17 d) was envisioned. First, the QA binds to the pore,slowing its departure from the vicinity of the channel. This increasesthe local effective concentration of the reactive electrophilic moiety,promoting covalent attachment to the channel protein, if it happens topossess a nucleophilic amino acid side chain at an appropriate distancefrom the QA binding site (˜20 Å away). Hence, the labeling of nativechannels by the photoswitch is promoted by the ligand bindinginteraction, as in classical affinity labeling (Johnson and Cantor,1977; Chowdry and Westheimer, 1979). In contrast to SPARK channels,where two components need to be added to cells (i.e. the Shaker channelgene and the MAQ photoswitch), this is a one-component system, whereonly PAL is required.

PALs were synthesized with several different reactive electrophiles,including epoxide, chloroacetamide, and acrylamide (FIG. 17 c). Theepoxide PAL (EAQ) was toxic to cultured neurons (see below). Thechloroacetamide PAL (CAQ) was less water-soluble than the acrylamide PAL(AAQ). Therefore, unless noted otherwise, AAQ was used for theexperiments described in this example.

FIG. 17A-D. The PAL Approach to Generating Light-Regulated Ion Channels.

(a) The photoswitch MAL-AZO-QA (MAQ) consists of photoisomerizableazobenzene (AZO) flanked by a quaternary ammonium (QA) group and acysteine-specific maleimide (MAL) reactive group. (b) MAQ reacts with K⁺channels that contain a genetically engineered cysteine located 20 Åaway from the pore allowing photocontrol of ionic current. In visiblelight (green), the photoswitch is extended, blocking ion conduction. InUV light (purple), AZO isomerizes to its cis form retracting the QA andallowing ion conduction. (c) PALs are identical to MAQ except that theycontain promiscuous electrophilic groups (red): acrylamide for AAQ,chloroacetamide for CAQ and epoxide for EAQ. (d) After the QA binds tothe pore, PALs react with nucleophiles endogenous to K⁺ channelsallowing regulation of native K⁺ channels with light.

PAL Imparts Light-Sensitivity on K⁺ Channels

The feasibility of the PAL approach was assessed, usingheterologously-expressed Shaker K⁺ channels as a test system. Additionof AAQ to a Shaker channel that has a cysteine-substitution (E422C)imparts light-sensitivity on the channel, just like MAQ (FIG. 18 a).However, AAQ imparts light-sensitivity to a similar extent on a Shakerchannel that does not have the cysteine substitution (E422; FIG. 2 b)and indeed, can confer light-sensitivity on a Shaker channel that iscompletely devoid of extracellular cysteines. The fraction of currentthat could be photoswitched was 39±1% for E422C and 48±2% for E422channels (FIG. 18 c), not significantly different from one another (n=6;p>0.3; Student's t-test). EAQ and CAQ also conferred light-sensitivityonto wild-type Shaker K⁺ channels. Hence PALs can find an attachmentsite at an appropriate distance from the pore, such that light-elicitedchanges in photoswitch length can allow or disallow block by the QAgroup.

To better understand how PAL imparts light-sensitivity on Shaker K⁺channels, it was attempted to identify the primary covalent attachmentsite relevant for light-dependent block and unblock. The structure ofthe conserved extracellular portion of a voltage-gated K⁺ channelprotein (Long et al., 2005) suggests that Shaker possesses at least 9accessible nucleophilic amino acid side chains that lie within 20 Å ofthe QA binding site. Mutagenesis of each of these sites suggests that noindividual one is essential for PAL to confer light sensitivity(Banghart et al., submitted).

If PALs are promiscuous in reacting with Shaker, perhaps they can alsoreact with other types of K⁺ channels that possess a QA binding site.This possibility was tested by expressing homomeric versions of Kv1.2,Kv1.4, Kv2.1, Kv3.1, Kv3.3, Kv4.2, and BKα channels in HEK cells (FIG.18 d). Whole-cell patch clamp recordings show that treatment with AAQimparted light-sensitivity on all of these channels, but some were moresensitive than others. Hence light turned on and off ˜80% of the currentthrough Kv1.4 and Kv4.2 channels, but only regulated ˜45% of the currentthrough BKα channels.

FIGS. 18A-D. Photocontrol of Wild-Type K⁺ Channels.

(a) AAQ photosensitizes Shaker channels that contain an engineeredcysteine (E422C). Current was elicited by stepping from −70 to +30 mVfor 200 msec. Visible light (green) blocks current through the channelswhereas UV light (purple) leads to unblock. (b) AAQ photosensitizeswild-type Shaker (E422). (c) Average percent photoswitching is similarfor Shaker with or without the engineered cysteine (n=6, p>0.3). (d) AAQsensitizes a variety of voltage-gated channels to light (n=6 for eachchannel).

PALs Impart Light-Sensitivity on Ion Channels in Cultured HippocampalNeurons

It was next tested whether PALs could impart light-sensitivity ontoendogenous K⁺ channels in neurons. FIG. 19 a shows steady-state I-Vcurves from a hippocampal neuron in culture, treated with 200 μM AAQ inthe dark for 15 min before recording. With this treatment, PAL should bein the extended trans configuration, and native channels blocked by PALshould unblock upon photoisomerization to the cis configuration.Consistent with this, exposure to 380 nm light increased thevoltage-gated outward current and 500 nm light reversed the effect.Voltage-gated outward current was photoswitched to a similar extent inneurons treated with 200 μM CAQ (not shown). In contrast, MAQ failed toimpart light sensitivity on outward current in untransfected neuronsthat express only their native ion channels (FIG. 19 b). The percent ofthe outward current that could be photoswitched was 63±8% (n=12) and2±5% (n=6) for AAQ- and MAQ-treated cells, respectively.

Although PALs confer light-sensitivity on many K⁺ channels, it wasexpected that other types of channels will be unaffected. External QA isoften used to help selectively remove K⁺ currents and reveal unalteredvoltage-gated Na⁺ and Ca²⁺ currents in neurons. Voltage-clamp recordingswith Cs⁺ in the patch pipette indicate that the transient inward currentdoes not become light-sensitive after treatment with PAL. Moreover,under current clamp, neither the rising phase nor the peak amplitude ofaction potentials is affected by light.

FIGS. 19A-C. Photocontrol of K⁺ Current in Cultured Hippocampal Neurons.

(a) Steady-state I-V curves for an AAQ-treated neuron illuminated withUV (purple) or visible (green) light. Illumination with visible lightblocks much of the outward current. (b) MAQ with its cysteine-specificreactive group does not sensitize native neuronal channels to light. (c)Average percent photoswitching of outward current in neurons treatedwith AAQ (n=12) and MAQ (n=6).

PALs Enable Optical Control of Action Potential Firing in CulturedHippocampal Neurons.

PAL treatment confers light-dependence on action potential firing. Undercurrent clamp conditions, 380 nm light turns off firing and 500 nm lightpromotes firing in neurons. The effects of light can be quite dramatic(FIG. 20 a), with neurons photoswitched between a rapid firing mode (10Hz) to complete quiescence within milliseconds of switching to 380 nmlight and back to repetitive firing with 500 nm light. Indeed, brief(100 msec) flashes of 500 nm light could be used to elicit actionpotentials on a one-to-one basis (FIG. 20 b).

FIGS. 20A and 20B. Regulation of Action Potential Firing with Light.

(a) Current clamp recording of a neuron treated with AAQ. Depolarizingcurrent was injected to induce continuous action potential firing invisible light (green). Illumination with UV light (purple) rapidlysuppresses action potential firing. High frequency firing resumes uponillumination with visible light. (b) One-to-one action potential firingupon exposure to short pulses of visible light.

PAL-modified K⁺ channels are blocked either in the dark or in visiblelight keeping the membrane potential tonically depolarized, which mightbe harmful to neurons. In addition, the reactive electrophile of PALcompounds might have deleterious effects on cells. To quantify thepossible toxicity of PALs, we used a fluorescent Live/Dead Assay (FIG.21). Cultured hippocampal neurons were first incubated for 15 or 60 minwith 200 μM of EAQ, AAQ, or CAQ. EAQ was highly toxic to neurons, evenwith a brief (15 min) treatment time, and was not used further in ourstudies. In contrast, AAQ and CAQ treatment resulted in the death of˜10% of neurons in 15 min, only slightly greater than followingtreatment with vehicle alone (DMSO; ˜5% in 60 min). Even with atreatment duration that was 4 times greater than needed to modify K⁺channels (60 min), AAQ and CAQ resulted in loss of only ˜15% of neurons.Additional experiments suggest that AAQ injection into the vitreoushumor of the rat eye has no detectable deleterious effects on retinalneurons. Hence at least for these cells and given our treatmentconditions, AAQ is not toxic to neurons in vitro or in vivo.

FIG. 21. Neuronal Survival after PAL Treatment.

Hippocampal neurons in culture were incubated with PAL for the indicatedtime and processed for a Live/Dead assay. Only EAQ resulted insubstantial toxicity.

PAL Imparts Light-Sensitivity on Neurons in Cerebellar Slices.

It was next tested whether PAL can regulate activity of freshly obtainedneurons in a neural circuit, using parasagittal cerebellar slicesobtained from young rats (P14-20). After pre-treating the slice with 200μM PAL for 10 minutes, a loose patch configuration was used to obtainextracellular recordings from cerebellar neurons (FIG. 22 a). Unlikewhole-cell recording, this configuration leaves the intracellular milieuintact and minimally perturbs neuronal activity, providing a rigoroustest of the effectiveness of the PAL technique under physiologicalconditions. It was found that full-field illumination of the slice with360 nm light increased firing frequency by 42±12% (n=5) in Purkinjeneurons, and illumination with 500 nm light restored the initial firingrate (FIG. 6 b). PAL-mediated sensitization to light was consistentlyobserved in every spontaneously firing neuron tested.

In principle, the observed change in Purkinje cell firing could resultfrom a change in excitatory synaptic input, a change in inhibitorysynaptic input, or a change in the intrinsic properties (i.e.voltage-gated ion channels) of the Purkinje cell itself. There may alsobe a combination of the above effects. To distinguish between thesepossibilities, the slice was incubated in pharmacological agents thatblock excitatory or inhibitory synaptic transmission. Addition of theAMPA-receptor antagonist DNQX failed to block light-triggered changes inactivity (FIG. 22 b). However, subsequent addition of gabazine, aselective GABA_(A) receptor antagonist, prevented illumination fromaltering the firing rate, consistent with light regulating the firing ofinhibitory neurons. Gabazine increased the basal firing rate of Purkinjecells up to ˜15 Hz, and it was considered that the blockade of the lightresponse might be due to gabazine-induced saturation of Purkinje cellfiring rather than regulation of inhibitory neuronal firing. However,Purkinje cells can fire at rates up to 40-80 Hz (Hausser and Clark,1997). Moreover, extracellular recordings from basket cells, the primarysource of inhibition to Purkinje neurons (REF), showed that 500 nm lightenhanced firing frequency by 324±160% (n=6), even in the presence of acocktail of neurotransmitter antagonists that block AMPA, NMDA, andGABA_(A) receptors (FIG. 22 c). Taken together, these results indicatethat the effects of light on Purkinje cell firing are mediated byinhibitory basket/stellate cells, which are preferentially sensitive toPAL.

FIGS. 22A-C. Photocontrol of Action Potential Firing in CerebellarSlices.

(a) Simplified circuit diagram of the cerebellum. + and − indicateexcitatory and inhibitory synapses. Abbreviations: granule cells, G;parallel fibers, pf; mossy fibers, mf; climbing fibers, cf. Recordingswere obtained from Purkinje neurons (PN) and interneurons(basket/stellate cells; B/S). (b) Extracellular loose patch recordingfrom a PN. UV light increased the frequency of action potentials in thePN. DNQX, which blocks excitatory synapses, failed to block the effectof light. Subsequent block of inhibitory synapses with gabazineabolished photocontrol, suggesting that PN photosensitivity is indirectand mediated by inhibitory neurons. (c) Extracellular loose-patchrecording from a B/S cell. AAQ renders B/S cells directlyphotosensitive. Photosensitivity remains after blocking excitatory andinhibitory synapses with DNQX, gabazine, and CPP (an NMDA receptorantagonist). UV light inhibits and 500 nm light restores B/S cellfiring. Light had no effect on untreated cells.

-   Banghart, M., Borges, K., Isacoff, E., Trauner, D. & Kramer, R. H.    (2004). Light-activated ion channels for remote control of neuronal    firing. Nat Neurosci 7, 1381-6.

REFERENCES

-   Bi A, Cui J, Ma Y P, Olshevskaya E, Pu M, Dizhoor A M, Pan Z H.    Ectopic expression of a microbial-type rhodopsin restores visual    responses in mice with photoreceptor degeneration. Neuron. 2006 Apr.    6; 50(1):23-33.-   Boyden E S, Zhang F, Bamberg E, Nagel G, Deisseroth K.    Millisecond-timescale, genetically targeted optical control of    neural activity.-   Nat. Neurosci. 2005 September; 8(9):1263-8.-   Kramer, R. H., Chambers, J. J., and Trauner, D. (2005).    Photochemical tools for remote control of ion channels in excitable    cells. Nature Chemical Biology 1:360-365-   Nagel G, Brauner M, Liewald J F, Adeishvili N, Bamberg E, Gottschalk    A.-   Light activation of channelrhodopsin-2 in excitable cells of    Caenorhabditis elegans triggers rapid behavioral responses. Curr    Biol. 2005 Dec. 20; 15(24):2279-84.-   Shoham S, O'Connor D H, Sarkisov D V, Wang S S. (2005). Rapid    neurotransmitter uncaging in spatially defined patterns. Nat.    Methods. 2:837-43.

Example 5 Remote Control of Neuronal Activity with a Light-GatedGlutamate Receptor

Methods

Photoswitch Synthesis and Generation of L439C Mutant of iGluR6.

Synthesis of MAG and introduction of cysteine L439C in iGluR6 werecarried out as described [11].

Cell Culture and Transfection.

HEK293 cells were plated at approximately 3×10⁶ cells/ml onpoly-L-lysine-coated glass coverslips and maintained in DMEM with 5%fetal bovine serum, 0.2 mg/ml streptomycin, and 200 U/ml penicillin at37° C. Cells were transiently transfected with various plasmids usinglipofectamine 2000 (Invitrogen). The amount of total transfectediGluR6(L439C) DNA and EYFP DNA per 2 ml well was fixed at 4 μg and 200ng, respectively. All recordings were carried out 36 to 48 hours aftertransfection.

Postnatal rat hippocampal neurons (P0-P5) were transfected by thecalcium phosphate method and recordings were carried out within 1-8 dayspost transfection. Neurons for patch-clamp experiments were transfectedwith either a fusion construct of GFP and iGluR6(439C) or co-transfectedwith iGluR6(L439C):YFP at a 3:1 ratio; for calcium imaging experimentsthey were co-transfected with iGluR6(L439C):DsRed at 3:1 ratio.

Conjugation of MAG Compounds.

To conjugate MAG to iGluR6(L439C), the compound was diluted to 10 μM inthe control solution (described below) from 1 mM stock solution in DMSO.The cells were incubated in the dark for 10-15 minutes. To conjugate MAGto iGluR6(L439C) in hippocampal neurons for patch-clamping experiments,the compound was diluted to 10 μM in a solution containing (in mM): 150NMDG-HCl, 3 KCl, 0.5 CaCl₂, 5 MgCl₂, 10 Hepes, and 5 glucose at pH 7.4.

Calcium Imaging and Spatially Selective Photoswitching

Imaging experiments were carried out on a Zeiss 510 META laser scanningmicroscope equipped with an Enterprise laser (Coherent) having 351 nmand 364 nm lines, and a 488 nm laser. The objective was a Zeiss PlanNeofluar 25×/0.80 Imm DIC (440542), and the dichroic mirror HFTUV/488/543/633 (both for 488 nm/364 nm photoswitching and Fluo-4imaging). Measurements with HEK cells were performed in a controlsolution containing (in mM): 135 NaCl, 5.4 KCl, 0.9 MgCl₂, 1.8 CaCl₂ and10 Hepes at pH 7.6. The control solution for hippocampal neurons was (inmM): 115 NaCl, 2.8 KCl, 1.0 MgCl₂, 1.0 CaCl₂, 10 glucose and 10 Hepes atpH 7.3. L-Glutamate and kainate were applied as reported in the text andfigures. Intracellular calcium was imaged with the Fluo-4 indicator(Molecular Probes, Invitrogen) excited at 488 nm and 4% laser power, andmeasuring emission between 495 nm and 527 nm using the META detector.Cells were conjugated during 10 minutes in 10 μM MAG in 0.3 mg/mlConcanavalin A type VI (Sigma), rinsed, and incubated in 10 μM Fluo-4 inthe dark for 1 hour. The experiments were then carried out within 1hour, to minimize Fluo-4 washout. Region-selective photoswitching wasobtained by alternating irradiation between 364 nm (3 iterations at 90%power) and 488 nm (3 iterations at 90% power) in selected regions, usingbuilt-in software intended for photobleaching experiments. The laserscanning microscope software was LSM 510 META version 3.2 SP2 (2003),and the MultiTime Series macro version 28-32 was used to control allcalcium imaging steps and photoswitching loops in a custom-made recipe.

HEK cells were transfected with iGluR6(L439C) about 48 hours prior toexperiments. No YFP cDNA was added in this case, to avoid the overlapwith Fluo-4 emission and to maintain a dark background. Expressing cellswere determined by brief responses to 300 μM glutamate, which allowedselection of the regions to be photoswitched. Neurons expressingiGluR6(L439C) were identified by co-transfection with DsRed at 3:1ratio.

No photobleaching was observed at the wavelengths and intensities usedin the experiments, which lasted less than 30 min. A significantreduction in calcium responses was evident ˜1 hour after loading thecells with Fluo-4, probably due to bleaching and/or washout of Fluo-4,which is to be expected with this non-ratiometric calcium dye. Althoughthe spatial resolution of photoswitching was not studied in detail inthese experiments, it is mostly dependent on the focusing of the 364 nmand 488 nm light and should be only limited by diffraction.

Calculation of calcium traces and time series image processing wascarried out with the microscope built-in software (physiology package)or with the ImageJ software version 1.33u (http://rsb.info.nih.gov/ij)with plugins LSM-Reader 3.2f and Z-profiler.

Whole-Cell Patch Clamping

Patch clamp recordings were carried out using an Axopatch 200A amplifierin the whole cell mode. HEK cells were voltage clamped at −60 mV andhippocampal neurons were current clamped at about −65 mV. Pipettes hadresistances of 2-5 MΩ and were filled with a solution containing, forHEK cells (in mM):145 CsCl, 5 EGTA, 0.5 CaCl₂, 1.0 MgCl₂ and 10 Hepes,pH7.2, and for neurons (in mM): 135 K-gluconate, 10 NaCl, 10 Hepes, 2MgCl₂, 2 MgATP, 1 EGTA, pH 7.4. The extracellular solution for HEK cellswas (in mM): 135 NaCl, 5.4 KCl, 0.9 MgCl₂, 1.8 CaCl₂ and 10 Hepes, pH7.6, and for hippocampal neurons: 138 NaCl, 1.5 KCl, 1.2 MgCl₂, 2.5CaCl₂, 10 glucose and 5 Hepes, pH 7.4. In order to block iGluR6desensitization, HEK cells were preincubated in 0.3 mg/ml concanavalin Atype VI (Sigma) [26, 27]. Illumination was applied using a TILLPhotonics Polychrome monochromator through the side port of the IX₇₀inverted microscope of the physiology rig (Olympus) and using either a40× or 60× objective. Fast photoswitching experiments were carried outwith a custom shuttered laser illumination setup mounted on a largebreadboard. Briefly, a 488 nm, 20 mW argon-ion laser (Laser Innovations)and a 374 nm, 8 mW Cube laser (Coherent) were combined with a dichroicmirror z405RDC (Chroma) and coupled into a P600-2-UV/vis optical fiber(Ocean Optics), using a 10×, 0.25 NA, 16.5 mm WD objective (Newport).The fiber was connected into the IX₇₀ microscope (Olympus) through theLaser B port using a custom-made adapter. Fast shutters (UniblitzUHS1T2-100 driven by VMM-T1 controllers, Vincent Associates) were placedat the output of each laser to control the illumination pulses viasoftware trigger.

Electrophysiological data was recorded with pClamp software, which wasalso used to automatically control the monochromator and laser shuttersby means of digital signals and sequencing keys.

Power Dependence of Photoswitching

Illumination power was reduced using a variable number of plastic slidesthat acted as neutral density filters. Current steps produced by 380 nmand 500 nm illumination were fitted by a single exponential function andthe normalized amplitude (A_(on), A_(off) respectively) and timeconstant (τ_(on), τ_(off) respectively) were obtained for eachillumination power. In order to calibrate irradiance (illuminationpower/area), the illumination power was measured with a Newport opticalpower meter model 840 at the exit of the microscope objective (OlympusUPlanApo 60×/1.2 W). The illuminated area was calculated from the imageof a calibrated grid (Zeiss).

Results

The chemical photo-switch MAG (consisting of: Maleimide for attachmentto an introduced cysteine, the photo-isomerizable Azobenzene moiety, andGlutamate as an agonist) was covalently attached to an introducedcysteine at residue 439 on the outer surface of the ligand-bindingdomain of the kainate receptor, iGluR6. As shown in Example 2, in thedark and under visible illumination (˜500 nm), MAG is mainly in itstrans form, with little or no activation of the receptor. Irradiation atlong wave UV (˜380 nm) induces cis photo-isomerization and positions theglutamate in the binding pocket, thus activating the receptor.Photoswitching of MAG leads to the opening and closing of thecation-selective pore of iGluR6(439C). Our goal here was to to determinethe properties of optical excitation of neurons expressing LiGlUR.

Spatial Control

One of the attractions of light-gated channels is that, in principle, itshould be possible to selectively activate only those cells that expressthem and fall within an illumination volume. Because iGluR6 can berendered highly calcium permeant with a single mutation [12], theability of light to activate calcium fluxes could be tested in singlecells by imaging intracellular calcium with a fluorescent indicator.Using a laser scanning confocal microscope equipped with 364 nm (UV) and488 nm (visible) lasers, spatially delimited photo-responses in selectcells were obtained. Perfusion of 300 μM glutamate elicited calciumresponses in all LiGluR expressing HEK293 cells. Single cells from amongthese were selected and irradiated them individually at 364 nm and 488nm. Optical stimulation triggered rises in calcium only in theilluminated cell. Similarly, light was used to stimulate hippocampalneurons expressing LiGluR with spatial selectivity. Calcium responses tophoto-stimulation was observed in single neurons that were doublytransfected with DsRed and iGluR6(439C) when the illumination waspointed at them. These results indicate that LiGluR can take fulladvantage of spatially delimited photo-stimulation [13], whereexcitation is confined to the illuminated volume because thephoto-responsive element is covalently attached to the channel. Theseexperiments provide a simple all-optical means of delineating neuralcircuits in culture or native tissue, using the LiGluR photo-switch as aremote actuator, standard fluorescent probes as sensors, andcommercially available laser scanning microscopes. More sophisticatedhardware platforms have also been developed recently [14] that shouldhelp improve temporal and spatial resolution and data processing.

Reproducible Bouts of Light-Evoked Firing in LiGluR Expressing Neurons

While calcium imaging could be used to detect the excitation ofLiGluR-expressing cells, patch clamping was used to characterize theexcitation quantitatively. The ability of light to excite culturedpostnatal hippocampal neurons, which were transfected with iGluR6(L439C)and FP and labeled with MAG, was examined. Current-clamp recordings wereperformed on the FP-positive neurons (FIG. 23 a).

Switching from 500 nm illumination, which deactivates the receptor, to380 nm to activate the receptor evoked large depolarizations and trainsof action potentials for periods defined by the duration at the shorterwavelength (FIG. 23 b). The amplitude of depolarization and thefrequency of action potential firing were reproducible (FIG. 23 b). Thetrains of light-evoked action potentials were similar to trains evokedby current injection. Light depolarized only the transfected neurons.Neurons in the same petrie dish that were not transfected did notrespond (FIG. 23 c), despite the fact that they had been exposed to MAGduring the labeling period. This lack of effect can be attributed to theabsence of a native cysteine in native GluRs at a location that wouldpermit MAG to attach and be in the correct geometry for its glutamateend to reach the binding pocket in either isomer of the azobenzene.

Photostimulation of the LiGluR-expressing neurons could be maintainedfor more than an hour in calcium imaging and for as long as seals heldin patch clamp (up to 45 minutes) without any indication of toxicity dueto illumination or MAG exposure. Cultured hippocampal neurons were oftenpatch-clamped two or more hours after MAG conjugation, indicating thatMAG is not toxic over a short period of time. Neuronal survivalfollowing 12 hours of continuous exposure to several concentrations ofMAG was also examined. This is much longer than the standard 15 minutelabeling time that was employed for the recordings. Staining for deadcells using a Live-Dead viability/cytotoxicity assay (Molecular Probes,kit L-7013) no difference was found in cell death between neuronsexposed to MAG and controls that were cultured in parallel (FIG. 23 d).These observations indicate that MAG has no detectable toxicity at theconcentrations in which it is employed. This is consistent with ourearlier observation that a model of MAG, which contains the(2S,4R)-4-substituted glutamate and a linker resembling half of theazobenzene tether, has an apparent affinity of 180 μM [Example 2]. Thus,the typical labeling concentration of 10 μM MAG will activate iGluR6only minimally. Activation of other iGluRs will be minimal becausesimilarly substituted glutamate analogues have been shown to beselective kainate receptor agonists.

FIGS. 23A-D. Photostimulation Yields Reproducible Trains of ActionPotential Firing.

(a) Hippocampal neurons transfected with LiGluR are easily identifiedfor patching by GFP fluorescence. (b) A neuron transfected withiGluR6(439C) and labeled with MAG is illuminated at 380 nm for hundredsof milliseconds to seconds, yielding reproducible depolarizations thattrigger trains of action potentials which fire at a frequency that ischaracteristic of the particular cell. Illumination at 500 nm turns theresponse off and permits repolarization. The firing pattern can besculpted by varying the duration of illumination at the two wavelengths.Illumination was with a monochromator. (c) Untransfected neuron has noresponse to light, despite exposure to MAG, but does fire repetitivelyin response to current injection. (d) MAG has no deleterious effect onneurons. Untransfected neurons incubated for 12 hours in MAG (as opposedto the standard labeling time of 15 min) showed no increase in celldeath compared to control.

Photoswitching in Milliseconds Generates Action Potentials and MockEPSPs

EPSPs mediated by native iGluRs are triggered by very brief (millisecondlong) and synchronous glutamate binding events at groups of receptors inpostsynaptic membranes. Ideally, an engineered system for triggeringneuron activity would operate on the same timescale. Because the rate ofactivation and deactivation of LiGluR depends linearly on illuminationintensity (FIG. 24 b), it was determined that millisecond switching waspossible under an illumination intensity of approximately 1 mW/mm². Thisintensity is typical of focused light from a standard fluorescence lampor monochromator system through standard objectives, as well as forsmall diode lasers. Indeed, brief (1-5 ms) pulses of light at −1 mW/mm²evoked currents that triggered reproducible patterns of actionpotentials (FIG. 25 a). Light-evoked patterns of firing were repeatablewithin a neuron and in different neurons. Furthermore, the amplitude ofthe responses could be easily attenuated with neutral density filters,in order to mimic EPSPs (FIG. 25 b, lower trace).

Rather than continuously illuminating the cells while switching back andforth between two wavelengths, we also evoked patterned action potentialfiring using only a brief pair of light pulses while otherwise keepingthe cell in the dark (FIG. 25 c). This allowed a significant reductionin light exposure (although, even under continuous illumination, nophoto-toxic effects were observed during any LiGluR experiments).

FIGS. 24A and 24B: Speed of Gating Depends on Intensity.

(a) Speed of activation and deactivation of iGluR6 current in HEK cellsunder voltage clamp increases with light intensity. (b) Rates of on andoff photoresponses in (a), measured from single exponential fits, areplotted against intensity, and yield a linear relation, which can beextrapolated to 1 KHz at −1 KW/m². This leads to the prediction that a 1mm region illuminated by a 1 mW laser should evoke significant currentsin ˜1 ms, which is of the timescale characteristic for synapticactivation of iGluRs. Illumination was with a monochromator.

FIGS. 25A-C. Designed Temporal Firing Patterns.

1-5 ms pulses of illumination are sufficient to significantly depolarizeneurons and to trigger single action potentials (APs). Scale bars=40 mVand 100 ms. (a) A train of 1 ms pulses of 374 nm light (laser) reliablytriggers the same temporal pattern of action potential firing in aneuron. Reproducible firing is triggered in two different neurons by thesame pattern of 374 nm light pulses. (b) In the same cell, a train of374 nm light pulses produces action potentials (top trace) or, when theillumination intensity is attenuated, sub-threshold EPSP-like responses(bottom trace). (c) LiGluR can be activated with a 2 ms pulse at 374 nmand deactivated with a 2-5 ms pulse at 488 nm to fire the neuron, andthe interval between action potentials can be in the dark to minimizeirradiation.

Because rapid stimulation of neurons is often employed in studies ofsynaptic plasticity, we were interested in determining the response ofneurons to light pulses delivered at high frequencies. We found that APsfollowed our optical stimulation reliably up to 30 Hz (FIG. 26), higherthan the frequency reported for ChR2 [Boyden et al., 2005]. We attributethis performance to the fact that LiGluR evokes larger currents, doesnot desensitize, and is capable of faster deactivation under opticaldrive (see Supplemental Material and Supplemental FIG. 1). In LiGluR,the loss of 1:1 firing at high frequencies appears to be a function ofthe firing properties of the neurons, not the kinetics of light-gating,since sub-threshold depolarizations were reliably evoked at allfrequencies tested (up to 100 Hz, data not shown).

FIG. 26. Neurons can Follow Photo-Stimulation of LiGluR Up to 30 Hz.

Trains of 5 ms laser pulses at 374 nm can reliably trigger actionpotentials up to a rate of 30 Hz. At 57 Hz only 12 action potentials areevoked by 20 stimuli, while at 86 Hz only 6 action potentials are evokedby 20 stimuli.

Light Evokes Depolarization and Action Potential Firing in a WavelengthDependent Manner

To characterize the amplitude of depolarization evoked by steadyillumination at different wavelengths iGluR6(439C) in HEK293 cellslabeled with MAG was examined under whole-cell current clamp. Channelopening by light evoked large steady-state depolarizations. By takingadvantage of the fact that the photo-stationary state of MAG (i.e. therelative proportion of azobenzene in cis and trans) can be preciselyvaried by illumination wavelength, it was possible to producesteady-state depolarizations whose amplitudes depended on wavelength(FIG. 27 a). Similar graded depolarizations could be evoked in culturedpostnatal hippocampal neurons that were transfected with iGluR6(439C)and exposed to MAG. As seen in the HEK cells, the amplitude ofdepolarization depended on wavelength, with a maximum at ˜380 nm. Thelargest depolarizations evoked a train of action potentials (FIG. 27 b).The wavelength dependence was used to adjust the size of EPSP-likewaveforms that were triggered by brief pulses of light, so that, forexample, pulses of light at 380 nm generated super-thresholddepolarizations and evoked action potentials, while EPSP-like responseswere induced in the same cell by pulses of light of the same duration,but at 430 nm (FIG. 27 c). Thus, the amplitude of brief excitatoryevents evoked by pulses of light can be controlled either by modifyingthe intensity of illumination at 380 nm, or by adjusting wavelength.

FIGS. 27A-C. Wavelength-Dependent Depolarization.

(a) In current-clamped

HEK cells expressing LiGluR, light induces depolarizations whoseamplitude depend on illumination wavelength. In this way, the cellmembrane potential can be accurately controlled across a wide range. (b)Illumination at a range of wavelengths depolarizes neurons. Somedepolarizations are large enough to reach threshold and trigger actionpotentials. (c) Patterned illumination with 380 nm light evokes actionpotentials while 430 nm light induces sub-threshold, EPSP-like responsesin the same cell. Illumination was with a monochromator.

Protracted Excitation in the Dark

We find that our azobenzene photoswitch is very robust, yieldingreproducible responses for many minutes in HEK293 cells under prolongedillumination [Volgraf et al., 2006]. Hippocampal neurons also toleratedtens of minutes of continuous illumination, alternating between 380 nmand 500 nm at intensities of 1 mW/mm² or more. These recordingstypically ended only upon loss of the seal, and recordings were equallystable with and without illumination. However, behavioral experimentsmay require activity to be manipulated over a much longer time scale,where photo-destruction of MAG or photo-toxicity to cells could become aconcern. To reduce this problem we explored the property of bi-stabilityof azobenzene in attempt to generate sustained trains of firing in thedark.

Depending on how azobenzene is derivatized, its higher energy cisconformation is stable for seconds to minutes in the dark [Pozhidaeva etal., 2004]. For MAG, the half-life is 17.65±0.03 minutes [Gorostiza etal., in preparation]. Thus, depolarization induced by a brief pulse of374 nm light is followed by sustained excitation in an ensuing periodduring which there is no illumination (FIG. 28, top). This sustainedexcitation in the dark can then be rapidly extinguished by a brief pulseof 488 nm light (FIG. 28, bottom). This molecular memory of MAG makes itpossible to trigger extended periods of excitation with minimalirradiation, during which time the cell fires at its characteristicfrequency. This is an advantageous property of LiGluR compared to ChR2.

FIG. 28. Brief Pulses of Illumination Followed by Dark Evoke SustainedFiring at Intrinsic Frequency of the Cell.

Due to the stability of the cis state of MAG, LiGluR activation by ashort pulse of 374 nm light yields a long-lasting depolarization thatcan trigger sustained trains of action potentials in the dark, which canbe then turned off with a short pulse of light at 488 nm. (top)Sustained excitation of hippocampal neurons occurs under continuous 374nm light (top trace) or when a brief pulse of 374 nm light is followedby darkness (bottom trace). (bottom) After a 5 ms pulse of 374 nm light,excitation is sustained for 10 seconds in the dark before being turnedoff by 488 nm light.

REFERENCES

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Example 6 Conferring Light Sensitivity to a Retina

Methods

Imaging Protocols

Flatmounts

Rats were sacrificed and the eyes were removed intact and placed inparaformaldehyde. After 1-2 hours the eyes were cut open, and the retinawas gently separated from the pigment epithelium. Small cuts were madearound the perimeter of the retina so that it can be flattened onto amicroscope slide. Images were taken with a large-field microscope (suchas a Zeiss Lumar Epifluorescence stereo microscope). The tissue wasilluminated with 488 nm light, which excites GFP. This lets one see thedistribution of our GFP-tagged channels throughout the entire retina.

Cryosections

Rats were sacrificed and the eyes are removed intact and placed inparaformaldehyde. After 1-2 hours the eyes were transferred to asaturated sucrose solution in order to dehydrate overnight. The nextmorning, eyes were imbedded in a mounting agent and rapidly frozen withdry ice. They were then sectioned in 10-20 μm slices using a standardcryostat. The sections were mounted on microscope slides and imaged on aconfocal microscope (such as the Zeiss 510 Meta UV/Vis confocal laserscanning microscope). The tissue was illuminated with 488 nm light,which excites GFP. This lets one see the distribution of our GFP-taggedchannels within individual cells.

Retcam

The Retcam is a digital fiber-optic camera developed for imaging retinaein vivo. Rats were anesthetized and laid flat on a lab bench. Their eyeswere dilated and then covered with a viscous gel, which acts as aninterface between the camera lens and the eye. The retina was imagedwith white light or with 488 nm light, which allows for the viewing ofGFP. This technique was used to monitor the expression of our GFP-taggedchannel without sacrificing the rat.

Physiology

Rats were sacrificed and the eyes were removed intact and placed in aNaCl-based saline solution. Eyes were then cut open in agelatin-bottomed petri dish, while being continually perfused withoxygenated saline. The retina was gently separated from the pigmentepithelium and cut into quadrants. These pieces were then incubated in apapain solution for 10-20 min at 37° C., transferred to an ovomucoidsolution (containing bovine serum albumin and trypsin inhibitor) for 5min, and a DNase solution for 10 min. The retina pieces were then rinsedand placed in a holding chamber containing oxygenated Ames medium untilused.

Prior to recording, each piece of retina was incubated in 100-300 μM AAQ(acrylamide-azobenzene-quaternary ammonium ion) for 30 min, removed,rinsed, then mounted flat in a recording chamber. Retinae werecontinually perfused with oxygenated saline during recordings, exceptwhen neurotransmitter blockers are used.

Recordings were done on an Olympus upright microscope. For dataacquisition, signals were amplified with an Axon Instruments amplifierand sent through an analog-to-digital converter to a computer runningClampEx (Axon Instruments). Signals were filtered at 2-5 kHz and sampledat 50 kHz. Illumination at 380 nm and 500 nm is provided by amonochrometer (Polychrome V, Till Photonics).

Micropipettes of borosilicate glass were pulled on a two-stage verticalpuller to obtain a resistance of 3-6 MΩ. These were filled with the samesaline as used in the bath. Loose-seal recordings are made by applyingfirm suction to the membrane of an identifiable retinal ganglion cell.During recording, all lights except that from the monochrometer wereturned off and the filter wheel was set to a 100% mirror to deflect thelight completely to the tissue. Recordings typically lasted 10-30 min.

ERGs

Rats were dark-adapted overnight, and the entire procedure was doneunder very dim red light. Rats were anesthetized and placed in astandard electroretinogram (ERG) setup. Recording electrodes wereembedded in contact-like cups, which were mounted onto each eye with aconductive adherent fluid. Reference electrodes were placed in themouth. A light source emitted brief flashes of white light at variousintensities. The electrical response of the retina to each light flashwas recorded; three trials per intensity were averaged together. Undernormal conditions, the ERG will consist of a small downward deflection(A-wave), which reflects the activity of photoreceptors, and a largerupward deflection (B-wave), which reflects the activity of bipolarcells.

Results

The data are presented in FIGS. 29-33. FIG. 29 depicts a retcam image ofan eye injected with a recombinant adeno-associated virus (rAAV) vectorcomprising a nucleotide sequence encoding a SPARK-GFP fusion proteinunder the control of a synapsin promoter (AAV-SYN-SPARK-GFP). FIG. 30depicts a flatmount of an eye injected with AAV-SYN-SPARK-GFP. FIG. 31depicts in vivo SPARK expression. FIG. 32 depicts data showing thatlight inhibits spontaneous firing of SPARK-expressing retinal ganglioncells in intact retina. Thus, the SPARK channel turns neurons intovirtual “off” cells.

FIG. 33A depicts extracellular recording from a PAL-treated retinalganglion cell showing optical control of firing. FIG. 33B depicts ERGrecordings from control (DMSO alone) and PAL-treated (DMSO+AAQ) rateyes, 7 days post injection. Thus, PAL imparts light sensitivity on ratretina without toxic consequences.

While the present invention has been described with reference to thespecific embodiments thereof, it should be understood by those skilledin the art that various changes may be made and equivalents may besubstituted without departing from the true spirit and scope of theinvention. In addition, many modifications may be made to adapt aparticular situation, material, composition of matter, process, processstep or steps, to the objective, spirit and scope of the presentinvention. All such modifications are intended to be within the scope ofthe claims appended hereto.

What is claimed is:
 1. An in vitro method of identifying an agent thatmodulates an activity of a non-light-regulated polypeptide, the methodcomprising: a) contacting a cell with a test agent which is a candidateagent for modulating the activity of the non-light-regulated polypeptidewhich is a calcium channel or a sodium channel, wherein the cellexpresses: i) the non-light-regulated polypeptide; and ii) alight-regulated polypeptide that regulates the activity of thenon-light-regulated polypeptide, wherein the light-regulated polypeptidecomprises a photoreactive synthetic regulator of protein function,wherein the photoreactive synthetic regulator comprises: a-i) a linkerdomain comprising a binding moiety that provides for stable associationwith covalent linkage to the light-regulated polypeptide, wherein thebinding moiety is other than a bromomethyl moiety; a-ii) aphotoisomerizable group; and a-iii) a ligand that binds to a ligandbinding site of the light-regulated polypeptide; b) exposing the cell tolight of a wavelength such that the ligand binds to a ligand bindingsite of the light-regulated polypeptide, wherein, in the absence of thetest agent, exposure of the cell to light results in activation of thenon-light-regulated polypeptide by the light-regulated polypeptide; andc) determining the effect, if any, of the test agent on the activity ofthe non-light-regulated polypeptide wherein a test agent that modulatesactivity of the non-light-regulated polypeptide following said lightexposure is a candidate agent for modulating the activity of thenon-light-regulated polypeptide.
 2. The method of claim 1, wherein thenon-light-regulated polypeptide is a sodium channel.
 3. The method ofclaim 2, wherein the non-light-regulated polypeptide is a calciumchannel.
 4. The method of claim 1, wherein step (c) comprises detectinga change in the intracellular concentration of an ion.
 5. The method ofclaim 1, wherein step (c) comprises detecting a change in the membranepotential of the cell.
 6. The method of claim 1, wherein the cell is amammalian cell.
 7. The method of claim 1, wherein the mammalian cell isa cell line.
 8. The method of claim 7, wherein the mammalian cell isselected from a 293 cell, a 3T3 cell, a COS cell, and a PC12 cell. 9.The method of claim 1, wherein the ligand-binding polypeptide isselected from an ion channel, a cation channel, a ligand-gated ionchannel, and a voltage-gated ion channel.
 10. The method of claim 1,wherein the ligand-binding polypeptide is a sodium channel, is apotassium channel, a chloride channel, a GABA receptor, or a serotoninreceptor.